Liposomal compositions and uses of same

ABSTRACT

Compositions comprising liposomes composed of whole cell membrane fraction are provided. The liposomes may be attached to, or encapsulate a pharmaceutical agent. Also provided are methods of generating and using these lipospmes.

RELATED APPLICATION/S

This application claims the benefit of priority under 35 USC 119(e) ofU.S. Provisional Patent Application No. 61/237,306 filed Aug. 27, 2009,the contents of which are incorporated herein by reference in theirentirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to liposomalcompositions and uses of same.

Liposome based DNA and drug delivery systems have been extensivelyinvestigated in the last four decades, and used as a mean to treatvarious conditions. Liposomal systems allow the efficient entrapment ofboth hydrophilic and hydrophobic compounds in a well-characterized,biocompatible and non-immunogenic lipid vesicle that can range fromnanometers to micrometers in diameter. Liposomes can also be targetedusing specific ligands such as protein conjugates or antibodies thatbind specific cellular receptors. In cancer therapy, liposomal systemsare of the most popular and well-investigated drug carriers. This ismainly due to the enhanced permeability and retention (EPR) effect,which refers to the increased vascular permeability of tumor vessels dueto tumor angiogenesis. The EPR effect results in the accumulation ofliposomes in the tumor extracellular fluid, which is exploited as apassive targeting mechanism. State of the art technologies in liposomaldrug delivery for cancer therapy primarily include drugs that areapproved for clinical use (e.g., DaunoXome™, Myocet™, Doxil™, Caelyx™).Several approaches are currently investigated for the targeting ofliposomal systems to cancer, which include the binding of targetingmoieties to the liposome surface (e.g., antibodies). Synthetic cationicliposomes are the most common vectors for DNA delivery although theircytotoxicity remains a concern irrespective of the preferred route ofDNA transfer both in vitro and in vivo. On the other hand, anionicliposomes that better resemble cell-derived liposomes (in term of theirelectric charge) were also shown to mediate gene transfer, but sufferfrom poor encapsulation efficiency due to the large size and thenegative charge of the uncondensed DNA. Improving encapsulationefficiency and protecting DNA from degradation was achieved bycomplexation of the DNA with cations or poly-cations that subsequentlyalso significantly improved the transfection efficiencies.

In the last decade several studies have revealed that certain primarycells, such as adult mesenchymal stem cells (MSC), adult hematopoieticstem cells (HSC) and endothelial cells, accumulate at tumormicroenvironments, when administered to tumor bearing animals. Recentdata suggests that isolated membrane fractions of tumor cells appear tocontain potent MSC attractants, more so than the cytoplasmic fractionsisolated from the same cells. This data implies that the mechanism ofMSC targeting to tumor cells is mainly governed by cell-to-cellinteractions via the binding of surface molecules found on tumors andMSC. However, cellular response to different soluble factors (i.e.,chemokines) secreted by angiogenic blood vessels and tumor cells issuggested to take some part in the MSC homing mechanism as well. Thehoming mechanism motivated studies on the use of these cells as atargeted delivery vehicle for cancer therapy. In these studies, primarycells were isolated and transduced with different genes of interest,either anti-cancer or reporter genes. The cells were transplanted totumor bearing animals and their homing to the tumor microenvironment wasdemonstrated using the expressed reporter proteins. Tumor inhibition wasachieved using the expressed anti-cancer proteins.

Liposomes, which are derived from the cytoplasmatic membrane ofmammalian cells, have been commonly used as a tool in the study ofmembranes and cellular mechanisms. Cell derived liposomes (CDL or CDLsin plural) have been also investigated as a tool for cancerimmunotherapy. In these studies, liposomes were prepared from themembranes of tumor cells and were used as adjuvant to evoke the immunesystem towards tumor antigens located on the liposome membrane. However,cell derived liposomes have never been produced from stem cells, norused as a delivery vehicle. Furthermore, no CDL system has ever beendeveloped as a targeting platform.

RELATED ART

-   Boone, C. W., Ford, L. E., Bond, H. E., Stuart, D. C. & Lorenz, D.    Isolation of plasma membrane fragments from HeLa cells. J Cell Biol    41, 378-392 (1969).-   Westerman and Jensen Methods Enzymol. 2003; 373:118-27.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a composition-of matter comprising a liposome attachedto, or encapsulating a pharmaceutical agent, the liposome being composedof a whole cell membrane fraction.

According to some embodiments of the invention, the cell is a humancell.

According to some embodiments of the invention, a cell source for thewhole cell membrane is selected from the group consisting of a stemcell, a primary cell, a cell-line, a non-tumorigenic cell, a cancer celland an immune cell.

According to an aspect of some embodiments of the present invention,there is provided a composition-of matter comprising a liposome composedof a whole cell membrane fraction of a stem cell.

According to some embodiments of the invention, the stem cell comprisesa human mesenchymal stem cell.

According to an aspect of some embodiments of the present inventionthere is provided a composition-of matter comprising a liposome composedof a whole cell membrane fraction of a primary human cell.

According to an aspect of some embodiments of the present inventionthere is provided a composition-of matter comprising a liposome composedof a whole cell membrane fraction of a non-tumorigenic human cell.

According to some embodiments of the invention, the cell membrane isgenetically modified to express an exogenous protein.

According to some embodiments of the invention, the exogenous protein isselected from the group consisting of a cell marker, a targeting moietyand the pharmaceutical agent.

According to some embodiments of the invention, the liposomeencapsulates, or attached to a pharmaceutical agent.

According to some embodiments of the invention, the pharmaceutical agentis a therapeutic agent.

According to some embodiments of the invention, thecomposition-of-matter is non-immunogenic in a human subject.

According to some embodiments of the invention, a cell source of thewhole cell membrane fraction comprises cells autologous to a hostsubject.

According to some embodiments of the invention, a cell source of thewhole cell membrane fraction comprises cells non-autologous to a hostsubject.

According to some embodiments of the invention, said liposome isattached to a synthetic polymer at an external surface thereof.

According to some embodiments of the invention, the pharmaceutical agentis a diagnostic agent.

According to some embodiments of the invention, the liposome isunilamellar.

According to some embodiments of the invention, the liposome is attachedto a synthetic polymer at an external surface thereof.

According to some embodiments of the invention, the synthetic polymer isa poly-ethylene-glycol (PEG).

According to some embodiments of the invention, the liposome has a sizerange of 30-1000 nm.

According to an aspect of some embodiments of the present inventionthere is provided a method of producing liposomes comprising,

-   (a) subjecting cells to hypotonic conditions, so as to obtain    ruptured cell membranes and/or ghosts; and-   (b) homogenizing the ruptured cell membranes and/or ghosts to    thereby produce liposomes.    -   According to some embodiments of the invention, the homogenizing        is effected by:-   (c) sonicating the ruptured cell membrane and/or ghosts; and    optionally-   (d) extruding the ruptured membrane and/or ghosts through a matrix    of pre-determined porosity.

According to some embodiments of the invention, the method furthercomprises conjugating a synthetic polymer to the liposomes followingstep (c).

According to an aspect of some embodiments of the present inventionthere is provided a method of encapsulating a pharmaceutical agent in aliposome, the method comprising making the liposomes according to themethod above and adding the pharmaceutical agent prior to the step ofhomogenizing.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as an activeingredient the composition-of-matter and a pharmaceutically acceptablecarrier.

According to an aspect of some embodiments of the present inventionthere is provided a method of delivering a pharmaceutical agent, themethod comprising administering to a subject in need thereof thecomposition of matter, thereby delivering the pharmaceutical agent.

According to some embodiments of the invention, the cell source of thewhole cell membrane fraction is autologous to the subject.

According to some embodiments of the invention, a cell source of thewhole cell membrane fraction is non-autologous to said subject.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying images and drawings.[1-10 images, 11 drawing]. With specific reference now to theimages/drawings in detail, it is stressed that the particulars shown areby way of example and for purposes of illustrative discussion ofembodiments of the invention. In this regard, the description taken withthe images/drawings makes apparent to those skilled in the art howembodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-C characterize human MSCs. Cell morphology as visualized byGiemsa staining (FIGS. 1A,B) and typical MSC (positive and negative)surface markers analyzed by flow cytometry (FIG. 1C).

FIG. 2 is a photograph showing the migration of hMSCs towards cancercells. DiI (red) labeled hMSC and DiO (green) labeled BHK, PC3, Cf2Thand COS-7 cells were drop-wise seeded. Maestro imaging following 72 hrsincubation demonstrated specific migration of the hMSCs towards PC3prostate cells while “avoiding” other cell-lines.

FIG. 3 is a graph showing targeting of MCF7 breast cancer cell-line byhMSCs stimulated by MCF7-derived condition media.

FIGS. 4A-B are Cryo-TEM images of cell-derived liposomes. Cell-derivedliposomes were prepared from the cytoplasmatic membranes of hMSCs andwere PEGylated by conjugation with monomethoxy-PEG. The resultingPEGylated (FIG. 4A) and un-PEGylated (FIG. 4B) CDLs were then imaged byCryo-TEM.

FIGS. 5A-C are graphs showing DLS and Zeta-Potential analysis of CDLs.Un-PEGyltated and PEGylated hMSC derived CDLs were analyzed for size,size distribution and charge by Number-weight DLS (FIG. 5A),Volume-Weight DLS (FIG. 5B) and Zeta-potential (FIG. 5C).

FIG. 6 shows the surface marker characterization of hMSCs derivedliposomes. CDL's were prepared from hMSCs, conjugated withTosyl-activated Dynabeads™ and analyzed by FACS for hMSCs specificmembranal markers (i.e., CD44, CD29, CD90 and CD105).

FIGS. 7A-B show the binding of CDLs prepared from hMSCs to prostatecancer cell-line (PC3). PC3 cells were labeled with DiO (green) andincubated with CDLs that were previously labeled with DiI (red).Cultures were imaged following 12 hrs incubation. Representative3D-projection (FIG. 7A) and single-slice (FIG. 7B) images are presented.

FIGS. 8A-B are graphs and FACS histograms showingconcentration-dependent binding of CDLs prepared from hMSCs to prostatecancer cell-line (PC3). PC3 cells were incubated with variousconcentrations of CDLs that were previously labeled with a redfluorescent dye (DiI). Following 24 hrs incubation, cells were washed,harvested and analyzed by FACS (FIG. 8A). The mean fluorescenceintensity of the cells was calculated and plotted vs. the naturallogarithm of CDLs concentration (FIG. 8B).

FIG. 9 show the specific binding of CDLs, prepared from conditionedhMSCs (i.e cell cultured with conditioned media of cancer cells), toprostate cancer cell-line (PC3). DiI-labeled CDLs were prepared fromhMSCs which were previously incubated for 24 hrs with condition mediaderived from a prostate cancer cell-line (PC3) and from a non-humancell-line (BHK). The resulting “conditioned” CDLs, as well as CDLsprepared from unconditioned hMSCs (control, NO CM), were incubated withPC3 and BHK cells for 15 min, 1 hr and 3 hrs. Following incubation, thecells were washed, harvested and analyzed by FACS. The percentage in themarker refers to the ratio of DiI-labeled cells within the marker. Thepercentage in brackets, designated on the upper-left histogram only,refers to the ratio of unlabeled cells within the marker. The marker andthe ratio of unlabeled cells within the marker are identical for allhistograms.

FIGS. 10A-B are cryo-TEM images of hMSCs derived liposomes entrappingsoluble Tumor necrosis factor-related apoptosis-inducing ligand(sTRAIL). sTRAIL-containing CDLs (FIG. 10A) and empty CDLs (FIG. 10B)were prepared at the same final concentration and imaged by Cryo-TEMunder the same conditions. To emphasize CDLs' content, the originalgrey-scale Cryo-TEM images (left pane) were re-colored to black andwhite (right pane).

FIG. 11 is a schematic illustration of the overall design of targetedcarriers based on cell-derived liposomes (CDL). Origin cells thatnaturally and specifically interact with target cells are selected as asource for cell derived liposomes. For example, MSC membranally interactwith cancer cells therefore are selected as a source for cancertargeting carriers. Source cells undergo hypotonic treatment to generateghost cells, which are then homogenized to produce CDL. Resulting CDLare then able to specifically bind their target cells in a similarmanner to the cells they are derived from.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to liposomalcompositions and uses of same.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

A major challenge facing cancer therapy is achieving a cytotoxic effecttowards cancer cells, while sparing the healthy ones. The importance ofthe development of novel targeted therapeutic delivery strategies forcancer therapy has long been recognized worldwide.

The present inventors have designed a novel delivery vehicle fortargeted delivery of therapeutic and diagnostic agents into cells andtissues. The delivery vehicle is liposome-based composed of a whole cellmembrane fraction which comprises both natural lipids and proteins. Byemploying native cell membranes, the delivery vehicles of the presentinvention may be formulated to be of low immunogenic potential, easilyhome to the target tissue and can be genetically modified to expresstherapeutic or targeting moieties.

As is illustrated below and in the examples section, which follows andfurther depicted in FIG. 12, the present inventors have generatedliposomes composed of whole cell membranes of mesenchymal stem cells,which are well-known for their homing capacities as well as theirimmuno-suppressive abilities (i.e., their ability to reduce inflammationand suppress immune cells) and hypo-immunogenic features (i.e.,stealth-like features that makes them less immunogenic and lessrecognizable as foreign matter when heterologously transplanted). Theliposomes exhibit the protein signature of mesenchymal stem cells and assuch are expected to mediate similar immunosuppression and migratoryproperties as intact mesenchymal stem cells. These cell derivedliposomes were further PEGylated to increase their bioavailability anddispersion and reduce their coagulation. The cell derived liposomes werealso treated to encapsulate a therapeutic agent. Altogether thesefindings, place the present delivery system as a pivotal tool in thediagnosis and treatment of human disease such as cancer.

Thus, according to an aspect of the present invention there is provideda composition-of-matter comprising a liposome attached to, orencapsulating a pharmaceutical agent, said liposome being composed of awhole cell membrane fraction.

As used herein the term “liposome” refers to fully closed carriermolecules comprising a spherical lipid membrane which itself is in aliquid crystalline phase or a liquid gel phase, in which an entrappedliquid volume is contained. The two liquid phases are immiscible. Thus,liposomes of the present invention (also referred to herein as cellderived liposomes (CDLs), similar to membranes of cells, are in anentirely gel/liquid state and/or liquid crystal state and not in a solidstate.

The liposomes of some embodiments of the present invention have anexpected protein to lipid ration of about 0.8 w/w.

Of note, the protein content of hMSCc CDLs is about 0.8 mg/10⁸ cells (asdetermined by Bradford assay). The lipid content can be easilydetermined using the Stewart phospholipids assay. It is expected to beabout 1 mg/10⁸ cells.

The following calcultation can be used to determine the theoreticalphospholipids content. Since the dry mass of a single mammalian cell isin the magnitude of 10⁻⁷ mg¹ and since phospholipids constitutesapproximately 10% of the dry cell mass² then the theoretical yield ofthe cell derived liposomes' production process (assumed 100% efficiency)should be in the magnitude of 10⁻⁸ mg phospholipids per single cell or 1mg per 10⁸ cells.

Liposomes include niosomes, transfersomes, emulsions, foams, micelles,liquid crystals, dispersions, lamellar layers and the like.

The liposomes may be unilamellar or multilamellar.

According to a specific embodiment of the invention, the liposomes areunilamellar, as determined by Cryo-TEM.

According to a specific embodiment of the invention, the liposomesexhibit native membrane symmetry and expression of native markers.

Liposomes of the present invention are composed of a whole cell membranefraction.

As used herein the phrase “cell membrane” or “cellular membrane” (whichmay be interchangeably used) refers to a biological membrane, whichsurrounds the cell or is an integral part of an organelle thereof (e.g.,chloroplast, ER, golgi, mitochondrion, vacuole, nucleus and a lysosome).

According to a specific embodiment of the present invention the cellmembrane refers to the plasma membrane. The use of plasma membrane is ofa specific advantage since it presents proteins, which are associatedwith cell-to-cell interactions, as well as other recognition molecules,such as receptors that bind soluble ligands.

As used herein “a whole cell membrane fraction” refers to a fraction,which does not include lipids alone but also includes membrane proteins.

Examples of membrane proteins include, but are not limited to, integralproteins, transmembrane proteins, lipid anchored proteins andglycoproteins.

According to an embodiment of the invention the whole cell membranefraction also includes carbohydrates.

According to a specific embodiment the cell is a eukaryotic cell [e.g.,mammalian (such as human), plant, insect cell].

According to an additional specific embodiment the eukaryotic cell is amammalian cell.

According to yet an additional embodiment the cell can be a primary cell(i.e., non-immortalized and at times not cultured) or a cell-line.

According to yet an additional embodiment the cell can be an embryoniccell.

Use of a primary cell may be advantageous for clinical use wherenon-cultured cells are used in autologous or non-autologous (syngeneicallogeneic or xenogeneic) settings.

According to a specific embodiment the eukaryotic cell is a stem cell.

As used herein, the phrase “stem cells” refers to cells, which arecapable of remaining in an undifferentiated state (e.g., pluripotent ormultipotent stem cells) for extended periods of time in culture untilinduced to differentiate into other cell types having a particular,specialized function (e.g., fully differentiated cells). Preferably, thephrase “stem cells” encompasses embryonic stem cells (ESCs), inducedpluripotent stem cells (iPS), adult stem cells, mesenchymal stem cellsand hematopoietic stem cells.

According to a specific embodiment the stem cell is a mesenchymal stemcell.

Mesenchymal stem cells are the formative pluripotent blast cells.Mesenchymal stem cells (MSCs) give rise to one or more mesenchymaltissues (e.g., adipose, osseous, cartilaginous, elastic and fibrousconnective tissues, myoblasts, cardiac like cells) as well as to tissuesother than those originating in the embryonic mesoderm (e.g., neuralcells) depending upon various influences from bioactive factors such ascytokines. MSCs can be isolated from embryonic yolk sac, placenta,umbilical cord, fetal and adolescent skin, blood, bone marrow, adiposeand other tissues, although their abundance in the bone marrow farexceeds their abundance in other tissues. MSCs have been shown to haveimmunosuppressive functions in various settings, including autoimmunediseases and transplantation, rendering liposomes generated therefromultimate tools in inflammatory and autoimmune settings.

Methods of isolating, purifying and expanding mesenchymal stem cells(MSCs) are known in the arts and include, for example, those disclosedby Caplan and Haynesworth in U.S. Pat. No. 5,486,359 and Jones E. A. etal., 2002, Isolation and characterization of bone marrow multipotentialmesenchymal progenitor cells, Arthritis Rheum. 46(12): 3349-60.

Preferably, mesenchymal stem cell cultures are generated by diluting BMaspirates (usually 20 ml) with equal volumes of Hank's balanced saltsolution (HBSS; GIBCO Laboratories, Grand Island, N.Y., USA) andlayering the diluted cells over about 10 ml of a Ficoll column(Ficoll-Paque; Pharmacia, Piscataway, N.J., USA). Following 30 minutesof centrifugation at 2,500×g, the mononuclear cell layer is removed fromthe interface and suspended in HBSS. Cells are then centrifuged at1,500×g for 15 minutes and resuspended in a complete medium (MEM, αmedium without deoxyribonucleotides or ribonucleotides; GIBCO); 20%fetal calf serum (FCS) derived from a lot selected for rapid growth ofMSCs (Atlanta Biologicals, Norcross, Ga.); 100 units/ml penicillin(GIBCO), 100 μg/ml streptomycin (GIBCO); and 2 mM L-glutamine (GIBCO).Resuspended cells are plated in about 25 ml of medium in a 10 cm culturedish (Corning Glass Works, Corning, N.Y.) and incubated at 37° C. with5% humidified CO₂. Following 24 hours in culture, nonadherent cells arediscarded, and the adherent cells are thoroughly washed twice withphosphate buffered saline (PBS). The medium is replaced with a freshcomplete medium every 3 or 4 days for about 14 days. Adherent cells arethen harvested with 0.25% trypsin and 1 mM EDTA (Trypsin/EDTA, GIBCO)for 5 min at 37° C., replated in a 6-cm plate and cultured for another14 days. Cells are then trypsinized and counted using a cell countingdevice such as for example, a hemocytometer (Hausser Scientific,Horsham, Pa.). Cultured cells are recovered by centrifugation andresuspended with 5% DMSO and 30% FCS at a concentration of 1 to 2×10⁶cells per ml. Aliquots of about 1 ml each are slowly frozen and storedin liquid nitrogen.

To expand the mesenchymal stem cell fraction, frozen cells are thawed at37° C., diluted with a complete medium and recovered by centrifugationto remove the DMSO. Cells are resuspended in a complete medium andplated at a concentration of about 5,000 cells/cm². Following 24 hoursin culture, nonadherent cells are removed and the adherent cells areharvested using Trypsin/EDTA, dissociated by passage through a narrowedPasteur pipette, and preferably replated at a density of about 1.5 toabout 3.0 cells/cm². Under these conditions, MSC cultures can grow forabout 50 population doublings and be expanded for about 2000 fold[Colter D C., et al. Rapid expansion of recycling stem cells in culturesof plastic-adherent cells from human bone marrow. Proc Natl Acad SciUSA. 97: 3213-3218, 2000].

MSC cultures utilized by the present invention preferably include threegroups of cells, which are defined by their morphological features:small and agranular cells (referred to as RS-1, herein below), small andgranular cells (referred to as RS-2, herein below) and large andmoderately granular cells (referred to as mature MSCs, herein below).The presence and concentration of such cells in culture can be assayedby identifying a presence or absence of various cell surface markers, byusing, for example, immunofluorescence, in situ hybridization, andactivity assays.

When MSCs are cultured under the culturing conditions of the presentinvention they exhibit negative staining for the hematopoietic stem cellmarkers CD34, CD11B, CD43 and CD45. A small fraction of cells (less than10%) are dimly positive for CD31 and/or CD38 markers. In addition,mature MSCs are dimly positive for the hematopoietic stem cell marker,CD117 (c-Kit), moderately positive for the osteogenic MSCs marker,Stro-1 [Simmons, P. J. & Torok-Storb, B. (1991). Blood 78, 5562] andpositive for the thymocytes and peripheral T lymphocytes marker, CD90(Thy-1). On the other hand, the RS-1 cells are negative for the CD117and Stro1 markers and are dimly positive for the CD90 marker, and theRS-2 cells are negative for all of these markers.

Other cells, which may be used as an effective source for whole cellmembrane fraction include, but are not limited to, endothelial cells,hepatic cells, pancreatic cells, bone cells, chondrocytes, neuronalcells and the like.

The cells can be used native (i.e., not manipulated by geneticmodification) or genetically modified to manipulate the membranecomposition of the cell.

The advantage of genetic modification is in its increased efficiency.Essentially all (>95%) the CDLs generated from genetically modifiedcells express the gene-of-interest. The gene-of-interest may beconstitutively expressed on the cell source (by integration to the cellsgenome) or transiently expressed (episomal expression) such as to avoidhazardous implications of stable transfection agents (e.g., lentiviraland retroviral vectors).

Thus, the cells may be genetically modified to express agene-of-interest (i.e., not naturally expressed in the native membranebut also in order to enhance the expression of endogenous proteins thatare naturally expressed on the cell's membrane but in lower levels).

According to specific embodiments, the gene-of-interest encodes amembrane protein. The gene-of-interest may be a native membrane proteinor modified to have a membrane localization signal and other motifsneeded for membrane anchorage e.g., a transmembrane domain.

Examples of membrane proteins which may be heterologously (exogenously)expressed include, but are not limited to, a targeting protein (e.g.,antibodies, receptors, membrane anchored ligands, decoys), a proteinwhich affects the chemistry of the membrane (e.g., structural proteins,charged proteins), a diagnostic protein (e.g., an enzyme as described inlength below) and a therapeutic protein (as described in length below).

A targeting moiety includes a targeting protein such as an antibody, areceptor ligand and a non-proteinecious molecule such as carbohydrates,which binds cell surface or extra-cellular matrix markers. For example,prostate-specific membrane antigen (PSMA) that is over-expressed onprostate cancer cells can be targeted by its ligand NAAG³ conjugated toa transmembranal motif (e.g, truncated LIME)⁴. This may be achieved, bygenetically engineering the cells (of which the CDLs are derived from)to express the chimeric or natural form of NAAG. For example, theexpression plasmid encoding LIME is constructed by PCR and subsequentinsertion of the corresponding fragment into pcDNA3.1 (Invitrogen). Theprimers also have BamHI (5′ primer and 3′ primer) site extension tofacilitate the subcloning. The PCR product is digested with BamHI andinserted into corresponding sites in pcDNA3.1(+) (CLONTECH Laboratories,Inc.). For expression vector encoding LIME-acetylaspartylglutamate(NAAG), the open reading frame can be inserted into plasmid coding LIMEsuch that the NAAG is conjugated trough its N-terminus and maintains itsC-terminus free to react with PSMA [i.e., LIME(C)—(N)NAAG-COOH]Alternatively, expression plasmid encoding NAAG-LIME chimera can beconstructed following the method described previously described forCD8-LIME chimera⁵. Fragments corresponding to NAAG and LIMEtransmembrane region were generated by PCR. Primers encoding the 3′sequences of the NAAG and the 5′ sequences of the LIME fragment weredesigned to overlap, such that annealing of the two products yielded ahybrid template. From this template, the chimera is amplified usingexternal primers containing XbaI sites. The NAAG-LIME chimera isinserted into pcDNA3.1(+).

As used herein, the phrase “surface marker”, refers to any chemicalstructure, which is specifically displayed at uniquely high density,and/or displayed in a unique configuration by a cell surface orextracellular matrix of the target cell/tissue.

For example, the targeting moiety may be useful for targeting to tumorcells. For example, it is generally accepted that the intracellularenvironment of tumor cells is more alkaline compared to their immediateextracellular environment, which in turn is more acidic than themicroenvironment found in the angiogenic blood vessels feeding thetumor. In addition, many previous studies have shown that the surfacecharges of tumor cells is more negative compared to benign normal cellsand even less invasive tumor cells. Accordingly, it may be useful toexpress membrane-bound enzymes and/or proteins, which will render theliposomes with a positive charge only in the acidic intermediateextracellular environment of the tumor. For example, any membranalprotein with a pI of about 7.2-7.4 that falls between the high alkalinepH of the angiogenic blood vessels (pH>7.4) and the low acidic pH of thetumor immediate extracellular environment (pH<7.2) can be used. Suchproteins can be specifically identified by cross referencing the RCSBProtein Data Bank (PDB) for human plasma membrane proteins. The expecteddesirable pI (7.2-7.4) for those proteins can be calculated using thestandard iterative algorithm that^(10, 11) that gives relatively preciseresults of pI calculations for raw protein sequences^(12, 13). Thealgorithm is used in the Compute pI/Mw tool at the ExPASy server. Suchliposomes are expected to have negative or neutral charge in thealkaline microenvironment of the angiogenic tumor vessels and positivecharge in the more acidic immediate extracellular environment of thetumor. Accordingly, this charge alteration will assist both liposomalextravasation, which is significantly enhanced for negative of neutralparticles, and intra-tumor delivery which is more easily accomplishedwith positively charge particles^(8, 14, 15).

Ample guidance regarding surface markers specifically over-expressed indiseases such as cancer, and antibodies specific for such surfacemarkers is provided in the literature of the art (for example, refer to:A M Scott, C Renner. “Tumour Antigens Recognised by Antibodies.” In:Encyclopedia of Life Sciences, Nature Publishing Group, Macmillan,London, UK, www.els.net, 2001).

Diseases associated with a target cell/tissue specifically displaying agrowth factor receptor/TAA surface marker which are amenable totreatment by the method of the present invention include, for example,some of the numerous diseases which specifically display growth factorreceptors/TAAs, such as EGF receptor, platelet derived growth factor(PDGF) receptor, insulin like growth factor receptor, vascularendothelial growth factor (VEGF) receptor, fibroblast growth factor(FGF) receptor, transferrin receptor, and folic acid receptor. Specificexamples of such diseases and the growth factor receptors/TAAs whichthese specifically display are listed in Table 1, below.

TABLE 1 Review reference Malignancy type Receptor* Kim, E. S. et al.,2001. Malignant glioma, glioblastoma, EGF Curr Opin Oncol 13, head andneck, breast, colon, receptor 506-13; Kuan et al., lung, prostate,kidney, 2000. Brain Tumor ovary, brain, pancreas, bladder Pathol. 2000;17: 71-8 George, D., 2001. Brain, prostate PDGF Semin Oncol 28, 27-33receptor Wang, Y., and Sun, Y., Breast, lung, colon, prostate IGFreceptor 2002. Curr Cancer Drug Targets 2, 191-207 Rosen, L. S., 2001.Solid tumors, acute and chronic VEGF Cancer J 7 Suppl 3, leukemias,myeloproliferative receptor S120-8; Giles, F. J., diseases, multiplemyeloma, 2001. Oncologist 6, 32-9 non-Hodgkin's lymphomas, and Hodgkin'sdisease Lappi, D. A., 1995. Melanoma, Caposi sarcoma, FGF receptor SeminCancer Biol 6, pancreas 279-88 Singh, M., 1999. Curr Leukemia, brain,colon, Transferrin Pharm Des 5, 443-51 kidney, bladder receptor*Abbreviations: EGF—epidermal growth factor, PDGF—platelet derivedgrowth factor, IGF—insulin like growth factor, VEGF—vascular endothelialgrowth factor, FGF—fibroblast growth factor.

In a preferred embodiment, the ligand is an antibody or an antibodyfragment, targeting antigens specific to a receptor on a target cell.Antibodies can be monoclonal antibodies, polyclonal antibodies orantibody fragments, which are target specific. In an embodiment, theantibodies attached to the liposomes are anti-CD19, anti-CD20, oranti-CD22, for specific binding to a B-cell epitope. These antibodies orantibody fragments are typically derived from hybridomas that showpositive reactivity toward the affected B-cells. It is contemplated thatother antibodies or antibody fragments targeting any other cell in thebody can be similarly used. For example, anti-CD19 antibodies are usedto target liposome containing an entrapped agent to malignant B-cells.The antibody recognizes a unique epitope, the CD19 surface antigen, onthe B-cells.

Methods of expressing heterologous proteins in eukaryotic cells are wellknown in the art.

Thus, an exogenous polynucleotide sequence designed and constructed toexpress at least a functional portion of the gene-of-interest may beexpressed in the cells from which membranes are later extracted.Accordingly, the exogenous polynucleotide sequence may be a DNA or RNAsequence of the gene-of-interest.

The phrase “functional portion” as used herein refers to part of theencoded protein (i.e., a polypeptide), which exhibits functionalproperties of the enzyme such as binding to a substrate. For example,the functional portion of an antibody may be the variable regionconferring specificity and additional/or alternatively the constantregion, i.e., Fc, which may activate complement and induce cell killing.For example, cells can be transfected with genes encoding one or moremembers from the GPCRs family (e.g., CCR5, CXCR4 etc.) that will renderthe liposomes targeted against abundant of cellular pathologiesincluding auto-immune and viral diseases (e.g., HIV/AIDS).

To express exogenous gene-of-interest in eukaryotic (e.g., mammalian)cells, a polynucleotide sequence encoding the gene-of-interest ispreferably ligated into a nucleic acid construct suitable for eukaryoticcell expression. Such a nucleic acid construct includes a promotersequence for directing transcription of the polynucleotide sequence inthe cell in a constitutive or inducible manner.

Constitutive promoters suitable for use for mammalian expression withthe present invention are promoter sequences, which are active undermost environmental conditions and most types of cells such as thecytomegalovirus (CMV) and Rous sarcoma virus (RSV). Inducible promoterssuitable for use with the present invention include for example theinducible promoter of the tetracycline-inducible promoter (Zabala M, etal., Cancer Res. 2004, 64(8): 2799-804).

The nucleic acid construct (also referred to herein as an “expressionvector”) of the present invention includes additional sequences, whichrender this vector suitable for replication and integration inprokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). Inaddition, a typical cloning vector may also contain a transcription andtranslation initiation sequence, transcription and translationterminator and a polyadenylation signal. By way of example, suchconstructs will typically include a 5′ LTR, a tRNA binding site, apackaging signal, an origin of second-strand DNA synthesis, and a 3′ LTRor a portion thereof.

The nucleic acid construct of the present invention typically includes asignal sequence for directing the translated polypeptide to the membraneand additionally a membrane anchor domain such as a transmembrane domainor a lipid based anchor (e.g., GPI).

Eukaryotic promoters typically contain two types of recognitionsequences, the TATA box and upstream promoter elements. The TATA box,located 25-30 base pairs upstream of the transcription initiation site,is thought to be involved in directing RNA polymerase to begin RNAsynthesis. The other upstream promoter elements determine the rate atwhich transcription is initiated.

Preferably, the promoter utilized by the nucleic acid construct of thepresent invention is active in the specific cell population transformed.Examples of cell type-specific and/or tissue-specific promoters includepromoters such as albumin that is liver specific [Pinkert et al., (1987)Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al.,(1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cellreceptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins;[Banerji et al. (1983) Cell 33729-740], neuron-specific promoters suchas the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad.Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al.(1985) Science 230:912-916] or mammary gland-specific promoters such asthe milk whey promoter (U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166).

Enhancer elements can stimulate transcription up to 1,000 fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream or upstream from the transcription initiation site.Many enhancer elements derived from viruses have a broad host range andare active in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for the present invention include thosederived from polyoma virus, human or murine cytomegalovirus (CMV), thelong term repeat from various retroviruses such as murine leukemiavirus, murine or Rous sarcoma virus and HIV. See, Enhancers andEukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor,N.Y. 1983, which is incorporated herein by reference.

In the construction of the expression vector, the promoter is preferablypositioned approximately the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

Polyadenylation sequences can also be added to the expression vector inorder to increase the efficiency of mRNA translation. Two distinctsequence elements are required for accurate and efficientpolyadenylation: GU or U rich sequences located downstream from thepolyadenylation site and a highly conserved sequence of six nucleotides,AAUAAA, located 11-30 nucleotides upstream. Termination andpolyadenylation signals that are suitable for the present inventioninclude those derived from SV40.

In addition to the elements already described, the expression vector ofthe present invention may typically contain other specialized elementsintended to increase the level of expression of cloned nucleic acids orto facilitate the identification of cells that carry the recombinantDNA. For example, a number of animal viruses contain DNA sequences thatpromote the extra chromosomal replication of the viral genome inpermissive cell types. Plasmids bearing these viral replicons arereplicated episomally as long as the appropriate factors are provided bygenes either carried on the plasmid or with the genome of the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryoticreplicon is present, then the vector is amplifiable in eukaryotic cellsusing the appropriate selectable marker. If the vector does not comprisea eukaryotic replicon, no episomal amplification is possible. Instead,the recombinant DNA integrates into the genome of the engineered cell,where the promoter directs expression of the desired nucleic acid.

The expression vector of the present invention can further includeadditional polynucleotide sequences that allow, for example, thetranslation of several proteins from a single mRNA such as an internalribosome entry site (IRES) and sequences for genomic integration of thepromoter-chimeric polypeptide.

Examples for mammalian expression vectors include, but are not limitedto, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1,pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as lentiviruses and retroviruses can be also used. SV40vectors include pSVT7 and pMT2. Vectors derived from bovine papillomavirus include pBV-1MTHA, and vectors derived from Epstein Bar virusinclude pHEBO, and p2O5. Other exemplary vectors include pMSG,pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vectorallowing expression of proteins under the direction of the SV-40 earlypromoter, SV-40 later promoter, metallothionein promoter, murine mammarytumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter,or other promoters shown effective for expression in eukaryotic cells.

Alternatively, cells, membranes, ghosts or CDLs (either of which may benative or genetically modified), may be chemically treated such as topresent a protein, a saccharide, a synthetic polymer, a peptide or anycombination of same. Methods of modifying the membrane with a syntheticpolymer are described herein below and in the examples section, whichfollows. Such a chemical attachment may be effected at any stage fromlive cultured or suspended cells to produced CDLs.

For example, the CDLs may be also chemically conjugated with folate thatmay further enhance their targeting and attachment to tumor cells, whichare known to express higher levels of folate receptors compared tobenign cells.

According to another example, it is also possible to permanentlymodulate the CDLs to have a more positive surface charge by treatingthem with cations, salts or polycations (e.g., Polybrene®,polyethyleneimine and Poly-L-Lysine) rendering them more positive tobetter target the tumor angiogenic vasculature.

Non-native material can be also introduced to the surface of the CDLs byfusion (e.g., PEG or detergent induced) with other liposomes (e.g.,cell-derived or synthetic) that may be comprised of well characterizedlipids, proteins and additives. Such fusion, creating hybrid CDLs, canbe used to conjugate any moieties (e.g., targeting, therapeutic,diagnostic, stealth-rendering etc.) to the CDLs and to alter theirsurface properties. See Example 5 for further guidance on liposomalfusion.

Synthetic polymers are typically used to prevent or reduce coagulation,increase dispersion, reduce interaction with blood components, evadenon-specific uptake by the mononuclear phagocytic system and prolong theparticle circulation time to a large extent thus, rendering theliposomes with properties and features that are commonly referred to asstealth properties or long-circulating liposomes. Accordingly, the pHnano-environment at the particle surface may also be dependent upon thelength of these molecules.

There are numerous polymers, which may be attached to lipids. Polymerstypically used as lipid modifiers include, without being limitedthereto: polyethylene glycol (PEG), polysialic acid, polylactic (alsotermed polylactide), polyglycolic acid (also termed polyglycolide),apolylactie-polyglycolic acid’ polyvinyl alcohol, polyvinylpyrrolidone,polymethoxazoline, polyethyloxazoline, polyllydroxyethyloxazolille,solyhydroxypryloxazoline, polyaspartarllide, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide,polyvinylmethylether, polyhydroxyethyl acrylate, derivatized cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose.

The polymers may be employed as homopolymers or as block or randomcopolymers.

The most commonly used and commercially available lipids derivatizedinto lipopolymers are those based on phosphatidyl ethanolamine (PE),usually distearylphosphatidylethanolamine (DSPE).

A specific family of lipopolymers, which may be employed by theinvention include PEG-DSPE (with different lengths of PEG chains) inwhich the PEG polymer is linked to the lipid via a carbamate linkage andPolyethyleneglycol distearoylglycerol. The PEG moiety headgrouppreferably has a molecular weight from about 750 Da to about 20,000 Da.More preferably, the molecular weight is from about 750 Da to about12,000 Da and most preferably between about 1,000 Da to about 5,000 Da.Two exemplary DSPE-PEG are those wherein PEG has a molecular weight of2000 Da, and of 5000a designated herein DSPE-PEG(2000) (DSPE-PEG2k) andDSPE-PEG(5000) (DSPE-PEG5k).

Specific families of lipopolymers, which may be also employed by theinvention, include C8 and C16 mPEG Ceramides (with different lengths ofPEG chains) in which the PEG-Ceramides contain ester linkages betweenthe PEG and ceramide moieties that allow the compound to be easilymetabolized. The PEG moiety headgroup preferably has a molecular weightfrom about 750 Da to about 2,000 Da. More preferably, the molecularweight is about 2,000 Da.

Conventional post-insertion PEGylation of common liposomes requiresheating or solublization in a detergent containing solution that mightdamage surface proteins and lead to encapsulate leakage. Therefore, CDLsmay be also PEGylated by the two following described methods or theircombination. Primarily, PEGylated CDLs will be prepared bydetergent-dialysis incorporation of PEGylated lipids into the ghost cellmembrane (prior to CDLs preparation). Following, direct PEGylation ofthe CDLs may be performed with monomethoxy-PEG activated by succinimidylsuccinate, which has been proven to increase the trasnfection efficiencyand reduce serum mediated inactivation of PEGylated lentiviralparticles, used as gene transduction vectors¹⁶.

Chemical binding of non-proteinaceous components (e.g., syntheticpolymers, carbohydrates and the like) to the liposomal surface may beemployed. Thus, a non-proteinaceous moiety, may be covalently ornon-covalently linked to, embedded or adsorbed onto the liposome usingany linking or binding method and/or any suitable chemical linker knownin the art. The exact type and chemical nature of such cross-linkers andcross linking methods is preferably adapted to the type of affinitygroup used and the nature of the liposome. Methods for binding oradsorbing or linking the enzyme and/or targeting moiety are also wellknown in the art.

For example, the enzyme and/or targeting moiety may be attached to agroup at the interface via, but not limited to, polar groups such asamino, SH, hydroxyl, aldehyde, formyl, carboxyl, His-tag or otherpolypeptides. In addition, the enzyme and/or targeting moiety may beattached via, but not limited to, active groups such as succinimidylsuccinate, cyanuric chloride, tosyl activated groups, imidazole groups,CNBr, NHS, Activated CH, ECH, EAH, Epoxy, Thiopropyl, Activated Thiol,etc. Moreover, the enzyme and/or targeting moiety may be attached via,but not limited to, hydrophobic bonds (Van Der Waals) or electrostaticinteractions that may or may not include cross-linking agents (e.g.,bivalent anions, poly-anions, poly-cations etc.).

Once the cell source is available the liposomes are made. Thus, there isprovided a method of producing liposomes comprising,

-   -   (a) subjecting cells to hypotonic conditions, so as to obtain        ruptured cell membranes and/or ghost cells (also termed ghosts);        and    -   (b) homogenizing the ruptured cell membranes and/or ghosts to        thereby produce liposomes.

The method may be practiced according to other well accepted protocolsknown in the art such as that of Boone, C. W., Ford, L. E., Bond, H. E.,Stuart, D. C. & Lorenz, D. Isolation of plasma membrane fragments fromHeLa cells. J Cell Biol 41, 378-392 (1969); and Westerman and JensenMethods Enzymol. 2003; 373:118-27 (each of which is incorporated hereinby reference) with or without modifications.

As used herein, the term “ghosts” refers to a cell that all of itscytoplasmic contents and/or nucleolus were removed by cell lysis and/ormembrane rapture so that only its outer cytoplasmatic/cell membraneremains; and

Without being bound to a specific protocol it is suggested in a specificembodiment that liposomes of the present invention are made in astep-wise manner. First, plasma membranes are isolated from cells (10⁹cells) primarily by using hypotonic treatment such that the cellruptures and ghost cells are formed. Altrnatively, ghost cells can beformed using mild sonication, freeze-thaw, French-press,needle-passaging or solublization in detergent-containing solutions.According to a specific embodiment hypotonic treatment is effected inTris-magnesium buffer (e.g., pH 7.4 or pH 8.6 at 4° C., pH adjustmentmade with HCl). Cell swelling is monitored by phase-contrast microscopy.Once the cells swell and ghosts are formed, the suspension is placed ina homogenizer. Typically, about 95% cell rupture is sufficient. Themembranes/ghosts are then placed in Sucrose (0.25 M or higher) forpreservation. To avoid adherence, the ghosts are placed in plastic tubesand centrifuged. A laminated pellet is produced in which the topmostlighter gray lamina consists only entirely of ghosts. However, theentire pellet is processed, to increase yields. Centrifugation (e.g.,3,000 rpm for 15 min at 4° C.) and washing (e.g., 20 volumes of Trismagnesium/TM-sucrose pH 7.4) may be repeated.

In the next step, the ghost fraction is separated by floatation in adiscontinuous sucrose density gradient. A small excess of supernatant isleft over the washed pellet, which now contains ghosts, nuclei, andincompletely ruptured whole cells. Additional 60% w/w sucrose in TM, pH8.6 is added to the suspension to give a reading of 45% sucrose on arefractometer. After this step, all solutions contain TM pH 8.6. 15 mlof suspension are placed in SW-25.2 cellulose nitrate tubes anddiscontinuous gradient is formed over the suspension by adding 15 mllayers, respectively, of 40% and 35% w/w sucrose, and then adding 5 mlof TM-sucrose (0.25 M). The material is now centrifuged at 20,000 rpmfor 10 min, 4° C. The nuclei sediment form a pellet, the incompletelyruptured whole cells are collect at the 40%-45% interface, and theghosts are collected at the 35%-40% interface. The ghosts are collectedand pooled.

In the next step, the ghosts are homogenized such as by sonication whichmay be followed by extrusion.

A specific sonication protocol relates to 5 second sonication using anMSE sonicator with microprobe at an amplitude setting of 8(Instrumentation Associates, N.Y.). This short period of sonication isenough to cause the plasma membrane of the ghosts to break up into cellderived liposomes (CDLs). Under these specific conditions organellemembranes are not disrupted and these are removed by centrifugation(3,000 rpm, 15 min 4° C.). Plasma membrane vesicles (CDLs) are thenpurified by sedimentation in a continuous sucrose density gradient.

Liposomes comprising one or more pharmaceutical agent of the presentinvention are preferably in the size range of 20-1000 nm e.g., 30-1000nm, 0.02-1.0 μm, more preferably 0.05-1.0 μm, more preferably 0.07-0.5μm and more preferably 0.1-0.3 μm. An advantage of liposomes smaller orabout 0.2 μm is that they can easily permeate through tumor vasculature(due to the EPR effect), they are not readily uptaken by macrophages andthey can undergo filter sterilization.

Extrusion of liposomes through a commercially available polycarbonatemembrane (e.g., from Sterlitech, Washington) or an asymmetric ceramicmembrane (e.g., Membralox), commercially available from Pall Execia,France is an effective method for reducing liposome sizes to arelatively well defined size distribution. Typically, the suspension iscycled through the membrane one or more times until the desired liposomesize distribution is achieved. The liposomes may be extruded throughsuccessively smaller pore membranes (e.g., 400 nm, 100 nm and/or 50 nmpore size) to achieve a gradual reduction in liposome size and uniformdistribution.

At any step prior to the homogenization, sonication and/or extrusion,that is, typically following ghosts preparation, a pharmaceutical agentmay be added to the reaction mixture such that the resultant liposomesencapsulate the pharmaceutical agent.

As used herein the phrase “pharmaceutical agent” refers to a therapeuticagent or diagnostic agent, which can be used to treat or diagnose amedical condition, respectively.

According to a specific embodiment, the composition comprising thepharmaceutical agent and the liposome is hypo or non-immunogenicespecially when the cell source is a mesenchymal stem cell.

Thus, the liposome of the present invention may have a pharmaceuticalagent adsorbed to a surface thereof or encapsulated therein eitherwithin the intra-liposomal polar phase or the lamellar non-polar lipidphase.

Methods of conjugating molecules (e.g., targeting moieties,pharmaceutical agents, synthetic polymers and the like) to liposomes arewell known in the art. For example, a the pharmaceutical agent (or anyother molecule) may be attached, conjugated or adsorbed to surface ofthe liposomes, ghosts or the cells of which the liposomes derive frombased on hydrophobic interactions (Van Der Waals bonds) or electrostaticinteractions with or without the use of cross-linking agents (e.g.anions and poly-anions). Hydrophobic and/or amphipathic pharmaceuticalagent (or any other hydrophobic and/or amphipathic molecule) may besoulibilized, partially soulibilized or partitioned into the cells,ghosts or liposomal lipid membranes with or without the use of detergentand/or by detergent dialysis. A pharmaceutical agent (or any othermolecule) may be attached, conjugated or adsorbed to surface of theliposomes, ghosts or the cells of which the liposomes derive from basedon covalent bonds with active groups. A pharmaceutical agent may beattached, conjugated or adsorbed to surface of the liposomes, ghosts orthe cells of which the liposomes derive from as a conjugate of anantibody or part of that specifically recognized a natural moiety foundon the liposomes, ghosts or cells. For example, pharmaceutical agent maybe adsorbed to the surface (inner or outer) of the liposomes via, butnot limited to, polar groups such as amino, SH, hydroxyl, aldehyde,formyl, carboxyl, His-tag or other polypeptides. In addition, thepharmaceutical agents may be adsorbed via, but not limited to, activegroups such as succinimidyl succinate, cyanuric chloride, tosylactivated groups, imidazole groups, CNBr, NHS, Activated CH, ECH, EAH,Epoxy, Thiopropyl, Activated Thiol, etc.

Entrapped in, adsorbed, expressed, conjugated, attached, and/orsolubilzed on the liposomes' surface or membrane is a therapeutic agentfor delivery to the target cells and/or tissues by one or more of, butnot limited to, the following mechanisms:

Direct intracellular delivery of the agent by means of membrane fusionbetween the liposomes and cells and/or liposomal uptake by endocytosis,phagocytosis or by any kind of transmembranal transport mechanism.

Diffusion and/or leakage of the agent from the liposome and consequentbinding to the surface of the target cells/tissue and/or uptake into thetarget cell/tissue by diffusion, endocytosis, phagocytosis or by anykind of transmembranal transport mechanism.

Binding to the surface of the target cells and/or tissues of an agentwhich is permanently, constantly or transiently expressed, attached,adsorbed, conjugated and/or solubilzed on the liposomes' surface ormembrane.

A variety of therapeutic agents can be entrapped in lipid vesicles,including water-soluble agents that can be stably encapsulated in theaqueous compartment of the liposome, lipophilic compounds that stablypartition in the lipid phase of the vesicles, or agents that can bestably or transiently attached, conjugated, adsorbed or expressed on tothe outer or inner surfaces of the liposomes, e.g., by electrostatic,covalent or hydrophobic interactions.

Exemplary water-soluble compounds include small molecules (i.e., lessthan 1000 Daltons) or large molecules (i.e., above 1000 Daltons);biomolecules (e.g. proteinaceous molecules, including, but not limitedto, peptide, polypeptide, post-translationally modified protein,antibodies etc.) or a nucleic acid molecule (e.g. double-stranded DNA,single-stranded DNA, ds/ss RNA (e.g., siRNA, antisense, ribozymes), ortriple helix nucleic acid molecules or chemicals. Therapeutic agents maybe natural products derived from any known organism (including, but notlimited to, animals, plants, bacteria, fungi, protista, or viruses) orfrom a library of synthetic molecules. Therapeutic agents can bemonomeric as well as polymeric compounds.

As mentioned above, the therapeutic agent may be a protein, such as anenzyme which compensates for loss in activity or poor expression of anendogenous enzyme e.g., the enzyme hexosaminidase A, a shortage of whichresults in Tay-Sachs disease.

Examples of therapeutic agents which may be delivered across a bloodbarrier to the brain, eye, testis or mammary gland include, but are notlimited to antibiotic agents, anti-neoplastic agents, anti-inflammatoryagents, antiparasitic agents, antifungal agents, antimycobacterialagents, antiviral agents, anticoagulant agents, radiotherapeutic agents,chemotherapeutic agents, cytotoxic agents, cytostatic agents,vasodilating agents, anti-oxidants, analeptic agents, anti-convulsantagents, antihistamine agents, neurotrophic agents, psychotherapeuticagents, anxiolytic sedative agents, stimulant agents, sedative agents,analgesic agents, anesthetic agents, birth control agents,neurotransmitter agents, neurotransmitter analog agents, scavengingagents and fertility-enhancing agents.

The liposome-entrapped compound may also be a diagnostic agent such asan imaging or a contrast agent as indium and technetium, enzymes such ashorseradish peroxidase and alkaline phosphatase, MRI contrast mediacontaining gadolinium, X-ray contrast media containing iodine,ultrasonography contrast media such as CO₂, europium derivatives,fluorescent substances such as carboxyfluorescein and illuminants suchas N-methylacrydium derivatives.

Once the liposomes are formed (i.e., with or without a pharmaceuticalagent), they may be characterized for their size distribution,composition, concentration, zeta potential, electrical surfacepotential, surface (local) pH, protein to lipid ratio and therapeuticefficacy in vitro and in vivo.

Experimentally tested liposomes of the present invention have thefollowing size values as described on Table 2 below:

TABLE 2 Without PEGylation: With PEGylation: Avg. by number 30 nm Avg.by number 100 nm Avg. by volume 200 nm Avg. by volume 215 nm Aggregationfactor: 200/30 = 7 Aggregation factor: 215/100 = 2

Empty liposomes or liposomes comprising one or more pharmaceutical agentof the present invention are preferably in the size range of 30-3000-nm,more preferably 50-500 nm, more preferably 30-300 nm, more preferably50-200 nm and more preferably 70-150 nm. An advantage of liposomessmaller or about 100-nm is its ability to penetrate through very narrowblood vessels which is of great significance in diagnostic andtreatment.

Any method known in the art can be used to determine the size of theliposome. For example, a Nicomp Submicron Particle Sizer (model 370,Nicomp, Santa Barabara, Calif.) utilizing laser light scattering can beused. Other methods of measuring liposome size include photocorrelationspectroscopy, laser diffraction, low-angle laser light scattering(LALLS), medium-angle laser light scattering (MALLS), light obscurationmethods (Coulter method, for example), rheology, or microscopy (light orelectron). The preferred average effective particle size depends onfactors such as the intended route of administration, formulation,solubility, toxicity and bioavailability of the compound.

Values of Zeta potential in experimentally tested liposomes are providedinfra. CDLs without PEGylation −17.9 to −15.5 mV.

With PEGylation (of ghosts-indirect): −13.2 mV. With PEGylation (ofCDls-direct): −10.2 mV.

Thus, liposomes of the present invention are characterized by a zetapotential of −20 to −15 mV without PEGylation and −15 to −10 mV withPEGylation.

As mentioned, liposomes of the present invention are advantageously usedin the clinic.

Thus, according to an aspect of the invention there is provided a methodof delivering a pharmaceutical agent, the method comprisingadministering to a subject in need thereof the above-describe liposome,wherein the pharmaceutical agent is enclosed therein or adsorbedthereon, thereby delivering the pharmaceutical agent.

According to an embodiment, the cells are target cells and the liposomescontain a targeting moiety, either chemically conjugated, heterologouslyadded, as described above, or natively presented in the membranes fromwhich the liposome is comprised (e.g., as in MSCs, which migrate totumor cells).

The cell source for the liposomes may be autologous or non-autologous(e.g., allogeneic, xenogeneic) to the subject.

The “target cell” referred to herein is a cell or a cluster of cells (ofhomogenous or heterogeneous population) and/or tissue to which asubstance is to be delivered by using the liposome. Examples thereofinclude cancer cells, vascular endothelial cells of angiogenic cancertissues, cancer stem cells, interstitial cells of cancer tissues, cellsaffected by genetic abnormality, cells infected by a pathogen and thelike. The “target molecule” may be any molecule presented the surface ofthe target cells or cells adjacent to the target cells. Another form ofthe target molecule includes molecules which are released from cells.Examples thereof includes extracellular matrix components, secretions orarchitectures of cancer cells or interstitial cells of cancer tissues,and specific examples thereof include tumor markers, structures betweencells and the like.

Delivering can be for diagnostic reasons (e.g., the liposome includes adiagnostic agent) or for treating (i.e., as a drug delivery tool,delivering a therapeutic agent).

The liposomes may be administered to the subject per se, or as part of apharmaceutical composition.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of the pharmaceutical composition is tofacilitate administration of the active ingredients to the subject.

Herein the term “active ingredient” refers to the therapeutic agent(with or without the liposome) accountable for the biological effect. Itis to be appreciated that the liposome per se may have immunomodulatoryfunction such as when prepared from membranes of MSCs or otherimmunomodulatory cells (e.g., immune B and T lymphocytes etc.). It isalso to be appreciated that the liposome per se may have a cytoxoiceffect on the target cells as due to membrane fusion with target cellsand consequent disruption to cell membrane, cytoskeleton and functions.In such a case measures are taken to include a targeting moiety suchthat the cytotoxic effect becomes specific.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to the subject and does not abrogate the biological activityand properties of the administered active ingredients. An adjuvant isincluded under these phrases.

Herein, the term “excipient” refers to an inert substance added to thepharmaceutical composition to further facilitate administration of anactive ingredient of the present invention or to increase shelf-lifestability. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and salts and types ofstarch, cellulose derivatives, gelatin, vegetable oils, EDTA, EGTA,Poly-L-Lysine, polyethyleneimine, Polybrene (hexadimethrine bromide),polyethylene glycols and other poly or single anions. The pharmaceuticalcomposition may advantageously take the form of foam, aerosol or a gel.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration include any of various suitablesystemic and/or local routes of administration.

Suitable routes of administration may, for example, include theinhalation, oral, buccal, rectal, transmucosal, topical, transdermal,intradermal, transnasal, intestinal and/or parenteral routes; theintramuscular, subcutaneous and/or intramedullary injection routes; theintrathecal, direct intraventricular, intravenous, intraperitoneal,intranasal, and/or intraocular injection routes, Catheterization with orwithout angio balloons; and/or the route of direct injection into atissue region of the subject.

The pharmaceutical composition may be manufactured by processes wellknown in the art, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active ingredients withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active ingredient doses.

Pharmaceutical compositions which can be used orally include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration via the inhalation route, the active ingredients foruse according to the present invention can be delivered in the form ofan aerosol/spray presentation from a pressurized pack or a nebulizerwith the use of a suitable propellant, e.g., a fluorochlorohydrocarbonsuch as dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane; carbon dioxide; or a volatile hydrocarbonsuch as butane, propane, isobutane, or mixtures thereof. In the case ofa pressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the active ingredients and a suitable powder base such as lactose orstarch.

The pharmaceutical composition may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

A pharmaceutical composition for parenteral administration may includean aqueous solution of the active ingredients in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredients may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition may also be formulated in rectalcompositions such as suppositories or retention enemas, using, e.g.,conventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical composition should contain the active ingredients inan amount effective to achieve disease treatment.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture and in vivo assays. For example, a dose can beformulated in animal models to achieve a desired concentration or titer.Such information can be used to more accurately determine useful dosesin humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma or brain levels of the active ingredients which are sufficient toachieve the desired therapeutic effect (minimal effective concentration,MEC). The MEC will vary for each preparation, but can be estimated fromin vitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of the composition to be administered will be dependent onthe subject being treated, the severity of the affliction, the manner ofadministration, the judgment of the prescribing physician, etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredients. Thepack may, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals there between.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”,W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Characterization of MSC Migratory and Targeting Abilities

Human MSCs were purchased from Lonza® (Switzerland) and characterizedusing Giemsa staining and FACS analysis for human mesenchymal stem cells(hMSCs) typical cell surface markers. As seen from FIGS. 1A-C the cellsappear to be positive for CD90, CD105, CD44, and CD29 and negative forCD133, CD31, CD34 and CD144, as expected for hMSCs.

The migratory abilities of the hMSCs towards cancer cells were tested aswell. For these experiments, hMSCs were labeled by a red-fluorescent dye(DiI) while several other cell lines (including a prostate cancercell-line—PC3) were labeled by a green fluorescent dye (DiO). Labeledcells were drop-wise seeded on tissue culture plates, incubated for 72hrs and imaged using the Maestro in vivo Imager (FIG. 2). As seen,specific migration of hMSCs towards PC3 cancer cells was demonstratedwhile “avoiding” interaction with other cell-lines (BHK, Cf2Th, andCOS-7).

Additional experiments were conducted to validate the targetingabilities of conditioned hMSCs to breast cancer cell-line MCF7. Forthat, human hMSCs were cultured with or without MCF7-derivedconditioning media, labeled with Dil (red) and co-cultured with MCF7cells labeled with DiO (green). Following 2 hr incubation, cultures werewashed and the coverage areas of each cell type and cell overlay(yellow) were determined using fluorescent microscopy image analysis.Assuming that the overlay of Dil and DiO is a consequence of physicalinteraction between the two cell types, the percent of overlay mayrepresent the amount of membranal interactions between the two celltypes. As can be seen from FIG. 3, the incubation of conditioned hMSCswith MCF7 cells resulted in 7% overlay, out of the total cell coveragearea, compared to no overlay when using unconditioned hMSCs (p<0.001).This apparent targeting evidently differs from the migration describedin FIG. 2 as it is mainly governed by membranal interactions between thehMSCs and the cancerous cells and not based on the migratory abilitiesof hMSCs that is largely mediated by soluble factors.

Example 2 Characterization of Cell Derived Liposomes Prepared from hMSCs

Cell Derived Liposome preparation—About 10⁷ Cells were harvested andwashed with PBS. Cells were then hypotonically treated by re-suspensionin ice cold Tris-magnesium (TM buffer, 0.01 M Tris, 0.001 M MgCl₂) pH7.4 for 15 min at 4° C. Following hypotonic treatment, the cells werehomogenized by rotor-stator mechanical homogenizer (IKA®, Taquara, RJ,Brazil) for 1 min at 22,000 rpm and turned into ghosts (95% rupturedcell membranes as confirmed by phase-contrast microscopy). Forstabilizing the ghosts' suspension, 60% (w/w) sucrose solution wasimmediately added to the suspension to make a final concentration of0.25M or 10% by volume. Ghosts were then centrifuged at 3000 rpm for 15min at 4° C. The supernatant was discarded and the pellet of ghosts wasthen washed twice with 0.25 M sucrose in TM-buffer pH 7.4, by repeatedsuspension and centrifugation at 3,000 rpm for 15 min at 4° C. In orderto create sonicated ghosts, the re-suspended pellet was then sonicatedfor 5 seconds at 27% amplitude using VibraCell VCX750 (Sonics &Materials Inc., Newtown, Conn.) and centrifuged at 3,000 rpm for 15 minat 4° C. The pellet of sonicated ghosts was then washed twice again with0.25 M sucrose in TM-buffer pH 8.6, by repeated suspension andcentrifugation at 3,000 rpm for 15 min at 4° C. For the formation ofunilamellar liposomes, the resuspeded pellet of sonicated ghosts wasmanually extruded by 21 successive passages trough polycarbonatemembranes with pore sizes of 0.4 μm and 0.1 μm (Osmonics Inc., MinnesotaUSA). The extruded liposomes were then centrifuged for 45 min at 150,000g at 4° C. The supernatant was discarded and the resulting liposomespellet was resuspended with TM buffer pH 8.6.

Cell derived liposomes surface protein PEGylation (according to themethof of Croyle, M. A. et al., 2004)—About 10⁷ Cells were harvested andwashed with PBS. Cells were then hypotonically treated by re-suspensionin ice cold Tris-magnesium (TM buffer, 0.01 M Tris, 0.001 M MgCl₂) pH7.4 for 15 min at 4° C. Following hypotonic treatment, the cells werehomogenized by rotor-stator mechanical homogenizer (IKA®, Taquara, RJ,Brazil) for 1 min at 22,000 rpm and turned into ghosts (95% rupturedcell membranes as confirmed by phase-contrast microscopy). Forstabilizing the ghosts' suspension, 60% (w/w) sucrose solution wasimmediately added to the suspension to make a final concentration of0.25M or 10% by volume. Ghosts were then centrifuged at 3000 rpm for 15min at 4° C. The supernatant was discarded and the pellet of ghosts wasthen washed twice with 0.25 M sucrose in TM-buffer pH 7.4, by repeatedsuspension and centrifugation at 3,000 rpm for 15 min at 4° C. In orderto create sonicated ghosts, the re-suspended pellet was then sonicatedfor 5 seconds at 27% amplitude using VibraCell VCX750 (Sonics &Materials Inc., Newtown, Conn.) and centrifuged at 3,000 rpm for 15 minat 4° C. The pellet of sonicated ghosts was then washed twice again with0.25 M sucrose in TM-buffer pH 8.6, by repeated suspension andcentrifugation at 3,000 rpm for 15 min at 4° C. For the formation ofunilamellar liposomes, the resuspeded pellet of sonicated ghosts wasmanually extruded by 21 successive passages trough polycarbonatemembranes with pore sizes of 0.4 μm and 0.1 μm (Osmonics Inc., MinnesotaUSA). The extruded liposomes were then centrifuged for 45 min at 150,000g at 4° C. The supernatant was discarded and the resulting liposomespellet was resuspended with TM buffer pH 8.6.

The protein content on the liposome's surface was determined using theBradford protein assay, referring to bovine serum albumin (BSA) asstandard. Succinimidyl succinate activated Monomethoxy-PEG was obtainedfrom Sigma Chemicals (St. Louis, Mo.) and was added to the resuspendedliposomes at a 10:1 ratio relative to the liposomes' protein content aspreviously determined by the Bradford assay. For example, 10 μg ofMonomethoxy-PEG were added for each 1 μg of protein. The conjugationreaction between the Monomethoxy-PEG and the liposomes was performed at25° C. with gentle agitation. The reaction was stopped by the additionof 10× L-lysine (Sigma Chemicals) with respect to the amount ofMonomethoxy-PEG added. Un-reacted free PEG, excess lysine, and reactionbyproducts were eliminated by buffer exchange over a Micro-Bio Spin P-30chromatography column (Bio-Rad) equilibrated with TM buffer pH 8.6.

CDL FACS analysis. Materials—Coupling Buffer—used for pre-washing andconjugating Dynabeads M-280 to the liposomes. The buffer was composed of0.1M Na-phosphate buffer pH 7.4, 2.62 g NaH₂PO₄×H₂O (MW 137.99) and14.42 g Na2HPO4×2H2O (MW 177.99) dissolved in distilled water andadjusted to 1 liter. Washing, blocking and Storage Buffer—PBS pH 7.4with 0.1% (w/v) BSA: Add 0.88 g NaCl (MW 58.4) and 0.1% (w/v) BSA to 80ml 0.01M Na-phosphate pH 7.4. Mix thoroughly and adjust volume to 100 mlwith 0.01M Na-phosphate pH 7.4.

Method: Liposomes were created form 2×10⁷ hMSCs as previously described.Tosyl-activated paramagnetic Dynabeads® M-280 (invitrogen) were used asthey were able to non-specifically and covalently bind any proteinand/or liposomes conjugated with proteins and to be later analyzed byflow-cytometry. Using magnetic separation device (MACS, Dynal™ MagneticParticle Separator—Invitrogen), the beads were washed with the couplingbuffer. To increase their ability to conjugate proteins, the beads werethen further washed with 3M ammonium sulfate added to the couplingBuffer. later, 4 samples were prepared containing 10⁷ beads each: Beadsonly, beads with liposomes, beads with liposomes to be labeled withsecondary antibody (isotype control) and beads conjugated with liposomesto be labeled with primary and secondary antibody (test sample). About5×10⁶ cell equivalent liposomes were added to each sample. Liposomes andbeads were then incubated for at least 12 hr at 4° C. After attachment,samples were re-suspended in the washing\blocking buffer. Each samplewas suspended in total volume of 200 μl. First, mouse MABs anti-humanCD29, CD44, CD90 or CD105 were added to the appropriate samples in aratio 1:100. Samples were incubated for 30 min in RT. Next, samples werewashed twice using the magnetic device. Then secondary ABs(FITC-conjugated goat anti mouse) were added and the samples wereincubated for 30 min at RT in the dark. All antibodies, primary andsecondary, were purchased from BD—Becton, Dickinson and Company.Following washing of the samples as mentioned before, the samples wererun and analyzed using FACSCalibur and CellQuest Pro (BD).

Results

PEGylated Cell-Derived Liposomes (PEG-CDLs) are expected to be protectedfrom opsonization and degradation, thus, having stealth properties andlonger circulation time in vivo. Also, PEGylation may reduce the risk ofnon-specific binding and fusion of liposomes as with non-targetcells¹⁷⁻¹⁹.

Cryo-TEM imaging of the CDLs demonstrated that the PEGylation had noapparent effect on the desirable small unilamellar morphology of theCDLs (FIGS. 4A-B). However, the PEGylated liposomes (FIG. 4A) seemedmore dispersed and less coagulated than the un-PEGylated liposomes (FIG.4B), that were imaged at the same concentration and under the sameconditions. Apparently, not only that the PEGylation does not damageliposomes' morphology but it may also improve their dispersion andstability. The size and size distribution of the CDLs were furtheranalyzed using number and volume weighing DLS analysis (Dynamic LightScattering, Malvern Nanosize). While number-weight DLS analysis (FIG.5A) demonstrated an increase in liposomes' size following PEGylation(from ˜30 nm to ˜100 nm), volume-weight DLS analysis (FIG. 5 b)demonstrated that the addition of PEG had a homogenizing effect on thesystem, exhibiting a significant reduction in the liposomes' sizedistribution. Evidently, the addition of PEG groups stabilized thesystem and prevented aggregation even though the Zeta-potentialdecreased from −17.9 mV to −10.2 mV (FIG. 5C).

Finally, the expression of MSC-specific surface markers, on the surfaceof hMSCs derived liposomes, was validated by FACS analysis (FIG. 6). Asseen, the CDLs retained their cytoplasmatic membrane symmetry and theexpression of correctly oriented typical hMSCs surface markers (i.e.,CD44, CD29, CD90 and CD105).

Example 3 Binding and Specific Targeting of Cancerous Cell-Lines by CDLs

Confocal microscopy imaging and flow cytometry analysis were used todetermine the binding of fluorescently labeled CDLs prepared from hMSCsto prostate cancer cells (PC3). As can be seen from FIG. 7A, mostvesicles favored cell binding. In addition, the vesicles were detectedinside and fused with the cell membranes (FIG. 7A).

Flow cytometry analysis demonstrates that most cells bind the vesicles(FIG. 8A) in a concentration-dependent manner (FIG. 8A), thus allowingto determine the extent of liposomal binding according to the cells'mean fluorescence intensity.

To test the specific targeting of cancerous cell-lines, DiI-labeled CDLswere prepared from hMSCs, which were previously incubated for 24 hrswith condition media derived from a prostate cancer cell-line (PC3) andfrom a non-human cell-line (BHK). The resulting “conditioned” CDLs, aswell as CDLs prepared from unconditioned hMSCs (control), were incubatedwith PC3 and BHK cells for 15 min, 1 hr and 3 hrs. Following incubation,cells were washed, harvested and analyzed by flow cytometry (FIG. 9).

The specificity index for every experiment, given a certain conditioningmedia

(NO CM, BHK-derived and PC3-derived) and incubation time (15 min, 1 hrand 3 hrs), was calculated according to the following equation:

$\begin{pmatrix}{Specificity} \\{index}\end{pmatrix} = \frac{\frac{\begin{matrix}{\% \mspace{14mu} {PC}\; 3\mspace{14mu} {cells}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {marker}} \\{{following}\mspace{14mu} {incubation}\mspace{14mu} {with}\mspace{14mu} {CDLs}}\end{matrix}}{\% \mspace{14mu} {PC}\; 3\mspace{14mu} {cells}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {marker}\mspace{14mu} {withot}\mspace{14mu} {CDLs}}}{\frac{\begin{matrix}{\% \mspace{14mu} {BHK}\mspace{14mu} {cells}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {marker}} \\{{following}\mspace{14mu} {incubation}\mspace{14mu} {with}\mspace{14mu} {CDLs}}\end{matrix}}{\% \mspace{14mu} {BHK}\mspace{14mu} {cells}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {marker}\mspace{14mu} {withot}\mspace{14mu} {CDLs}}}$

The specificity index results, summarized in Table 3 below, not onlyillustrates that the system exhibits specificity towards cancerous cellsbut that this specificity, as excepted, decreases with incubation time.In addition, the specificity index values show that the system'sspecific affinity towards cancer cells can be largely affected bysubjecting the cells to various conditioning media prior to CDLspreparation.

TABLE 3 Specificity index of CDLs binding to prostate cancer PC3cell-line CDL incubation time with PC3 hMSCs Conditioning and BHK cellsmedia 15 min 1 hr 3 hr No CM 1.91 1.50 1.25 BHK derived 3.45 3.43 1.10PC3 derived 2.92 2.31 1.50

Example 4 Protein Entrapment within CDLs

sTRAIL Production

Mediums and buffers—1 L 2YT medium was prepared from 16 gr Bacto™Tryptone (BD number 211705), 10 gr Bacto™ Yeast Extract (DIFCO number212750) and 5 gr NaCl (Chemically Pure). Medium used for culturing inPetri dished contained 16 gr Agar Granulated (DIFCO number 214530) ontop of the above components. The medium was autoclaved for sterility.PBSX10 was prepred from 2 gr KCl, 2.4 gr KH₂PO₄, 14.4 gr Na₂HPO₄.7H₂Oand 80 gr NaCl. Volume was adjusted to 1 L with DDW and the buffer wasfilter sterilized through 0.2 μm filter.

Plasmids, DNA, bacteria and antibiotics—GST-sTRAIL coding DNA was kindlysupplied by Dr. Stanley Lipkowitz, Bethesda, Md. in pGEX-2TK plasmidintroduced into E. coli BL21 using Ampicillin 100 μg/ml as a selectionagent. Ampicillin stock was prepared from ampicillin Sodium Salt (Sigmanumber A9518) dissolved in Ultra Pure DDW (UP-water) to a finalconcentration of 100 mg/ml and filtered through 0.2 μm filters.

Additional materials: Ethyl Alcohol 99% Dehydrated (FRUTAROM number2355516400); D(+)GLUCOSE (Sigma number G5146); IPTG (Ornat Biochemicalsnumber INA-1758-1400); Complete Mini EDTA-Free Protease inhibitorcocktail tablets (Roche Applied Science number 04693159001);DTT—DL-Dithiothereitol solution (Sigma number 43816); GSH BEADS (GEHealthcare); Glutathione Sepharose 4B (10 ml, Danyel Biotech number17-0756-01); and L-Glutathione-reduced (Sigma number G4251).

Equipment—Amicon Ultra-15 centrifugal filters (Millipore numberUFC901024); and French Press cell disruption system. All solutions werefiltered for sterility through a 0.2 μm filters; all procedures up toDay 2 (step 4, pellet of bacteria after IPTG O/N induction) were carriedout in a Sterile Hood.

Swith 1/100 GST-TRAIL glycerol stock (e.g., 400 μl GST-trail glycerolstock in 40 ml medium). The solution was incubated O/N 37° C. in ashaking incubator at 250 RPM.

Day 2

STEP 2: The “starter” culture was spun down at 1000 g for 15 min toremove the antibiotics. The supernatant was discarded and resuspended in40 ml of fresh 2YT. In a Sterile Hood, the resuspended 40 ml of the O/Npreparation from step number 1 was added to 2 L of 2YT in a 4 L flask(alternatively add the resuspended pellet of 20 ml of the O/Npreparation from step number 1 to two 2 L flasks each containing 1 L of2YT). The solution was incubated for 2-3 hours in a 37° C. shakingincubator at 250 RPM. Measures are taken not incubate for more than 3hours until O.D. is 2.5-3.0 (it is recommended to measure O.D.₅₉₅ after2 hours).

STEP 3: Just before IPTG induction, PBS was added to a finalconcentration of 0.1× to maintain the pH of the culture. EtOH (99%Dehydrated) was added to a final concentration of 2% (40 ml in 2 Lculture) to increase the solubility of the protein. 10 ml/L of 0.5MGlucose was added as a carbon source to a final concentration of 5 mM.

STEP 4: 500 μM IPTG were added to the supplemented culture. The culturewas incubated over night in a shaking incubator (250 RPM) at 20-25° C.

Day 3

STEP 5: Bacteria was pelleted at 6,000 g for 10 min and the supernatantwas discarded. All bacteria were resuspended in a 50 ml Falcon Tubeusing 40 ml PBS supplemented with 4 protease inhibitor tablets (RocheApplied Science) 1 tablet per 10 ml PBS.

STEP 6: Cells were lyzed by running the bacteria from step number 5twice through a French Press cell disruption system. Alternatively, 10ml aliquots in 50 ml tubes were sonicated on ice at 30% power by 4bursts of 10 sec each. After Cell disruption, the following was added toeach 40 ml of cell lysate: 0.1% Triton-X (40 μl of TritonX100), 1 mMMgCl₂ (40 μl of 1M stock MgCl₂) and 1 mM DTT (40 μl of 1M stock DTT).The solution was mixed thoroughly and incubated at RT for 15 min on arocker or shaker.

STEP 7: The bacterial lysate was spun down for 10 min at 16,900 g and 4°C. Supernatants were aspirated and collected in 50 ml tubes.

STEP 8: Binding to GSH (Glutathione—Sepharose 4B Beads)—In a 15 mlFalcon Tube, 3 ml of GSH Beads were washed three times with PBS.Collected supernatant was centrifuged again because of mass bead loss.The washed beads were added to the bacterial cell lysate from stepnumber 7 and incubated with tumbling for 1 hour at 4° C.

STEP 9: The bacterial cell lysate, containing the sepharose beads fromstep number 8, was spun down at 2000 RPM for 1 min in a MULTI CENTRIFUGECM 6M ELMI to separate the protein-conjugated beads from thecell-lysate. The supernatant was collected and was centrifuged again topellet the remaining sepharose beads in the supernatant (that might havenot pelleted during the first centrifugation). The pellet from bothcentrifugations, containing the sTRAIL-conjugated beads, was washed 5times with 5 ml of PBS supplemented with 0.1% Triton-X100, 150 mM NaCland 1 proteinase inhibitor tablets per 20 ml PBS.

STEP 10: Elution of GST-sTRAIL—The beads were spun down as before andthe supernatant was aspirated. 3 ml of 50 mM Glutathione (pH 8.5) in 10mM Tris-HCl and 100 mM NaCl were added. Each 3 ml was vortexed for 2 minand the protein was eluted into the supernatant. The supernatant wasaspirated as before and the supernatant kept. The procedure of elutionwas repeated 3-4 times.

STEP 11: The protein was concentrated using Amicon Ultra-15 10KNMWLnumber UFC9010, giving a protein yield of about 5 mg/L culture.sTRAIL was produced at a final concentration of 0.2 mg/ml

sTRAIL entrapment—About 10⁷ Cells were harvested and washed with PBS.Cells were then hypotonically treated by re-suspension in ice coldTris-magnesium (TM buffer, 0.01 M Tris, 0.001 M MgCl₂) pH 7.4 for 15 minat 4° C. Following hypotonic treatment, the cells were homogenized byrotor-stator mechanical homogenizer (IKA®, Taquara, RJ, Brazil) for 1min at 22,000 rpm and turned into ghosts (95% ruptured cell membranes asconfirmed by phase-contrast microscopy). For stabilizing the ghosts'suspension, 60% (w/w) sucrose solution was immediately added to thesuspension to make a final concentration of 0.25M or 10% by volume.Ghosts were then centrifuged at 3000 rpm for 15 min at 4° C. Thesupernatant was discarded and the pellet of ghosts was then washed twicewith 0.25 M sucrose in TM-buffer pH 7.4, by repeated suspension andcentrifugation at 3,000 rpm for 15 min at 4° C. In order to createsonicated ghosts, the re-suspended pellet was then sonicated for 5seconds at 27% amplitude using VibraCell VCX750 (Sonics & MaterialsInc., Newtown, Conn.) and centrifuged at 3,000 rpm for 15 min at 4° C.The pellet of sonicated ghosts was then washed twice again with 0.25 Msucrose in TM-buffer pH 8.6, by repeated suspension and centrifugationat 3,000 rpm for 15 min at 4° C. After, sTRAIL was added to thesuspended sonicated ghosts (in TM buffer pH 8.6) to a finalconcentration of 1 μg per 1 ml of ghost suspension. For the formation ofunilamellar liposomes containing sTRAIL, the sTRAIL-containingresuspeded pellet of sonicated ghosts was manually extruded by 21successive passages trough polycarbonate membranes with pore sizes of0.4 μm and 0.1 μm (Osmonics Inc., Minnesota USA). The extruded liposomescontaining sTRAIL were then centrifuged for 45 min at 150,000 g at 4° C.The supernatant containing excess non-encapsulated sTRAIL was discardedand the resulting liposomes pellet was resuspended with TM buffer pH8.6.

Results

TRAIL—tumor necrosis factor-related apoptosis-inducing agent is a typeII transmembrane protein that induces apoptosis in tumor cells ofdiverse origins, while sparing most normal cells²⁰⁻²⁴. Delivery of bothfull length and truncated, secreted forms of TRAIL (sTRAIL) were shownto induce apoptosis in a variety of cancer cells both in culture and invivo^(25, 26). Our preliminary experiments with sTRAIL included itsproduction and passive encapsulation within hMSCs CDLs at a finalconcentration of 1 μg/ml. Cryo-TEM imaging of the resultingsTRAIL-containing CDLs (FIG. 10A left pane), compared to empty CDLs(FIG. 10B, left pane) prepared and imaged under the same conditions,demonstrates the accumulation of 14-20 nm protein micelles within theCDLs. The sTRAIL micelles are even more clearly visible after digitallyre-coloring the images from grey-scale to black-and-white (FIGS. 10A and10B, right panel).

Example 5 Preparation of “Hybrid CDLs” by Fusion with Other Liposomes

Various molecules (e.g., proteins, lipids, additives and evenencapsulates) can be introduced, conjugated or attached onto the surfaceof the CDLs by means of fusion between the CDLs and other liposomes(synthetic or cell-derive), thus creating—“Hybrid CDLs”. For example, aliposomal formulation made from synthetic well characterized lipids maybe conjugated with a protein on its surface or may contain a desirableencapsulate. Then, by means of induced membrane fusion between the saidsynthetic liposomes and CDLs a hybrid CDL may be formed. These hybridCDLs contain both lipids and proteins from the cell-membrane they derivefrom and the lipids and proteins that were originally formulated on thefused synthetic liposomes. Such introduction of ‘non-native’ materialsonto the Hybrid CDLs may be used to attach or conjugate any molecule ormoieties related, but not limited to, liposomal targeting, therapeuticeffect, diagnostic effect, stealth-rendering properties etc. Such fusionmay be also used to change the biochemical or chemophysical propertiesof the CDLs membranes by introduction of synthetic lipids, additives(e.g., cholesterol, ceramides) etc. Such fusion may be also used toincrease the encapsulation efficiency in the said CDLs. Sinceencapsulation in CDLs is mainly limited to passive encapsulation, fusionwith synthetic liposomes that were actively loaded with highconcentration of encapsulates may significantly improve the CDLs'encapsulation efficiency.

Methods for preparation of synthetic liposomes are well known in the artand mainly include hydration of dehydrated lipids to form lamellarstructures and consequent homogenization of those lamellar structures tocreate liposomes. Synthetic liposomes of the said application can beproduced by any method known in the art including, but not limited to,solvent evaporation, solvent replacement, detergent dialysis, extrusion,sonication, freeze-drying, reverse phase evaporation, ethanol/etherinjection, agitation and/or any other form of mechanical homogenization.Liposomes can be prepared from a variety of synthetic and naturallyderived lipids and may or may not contain additional additives (e.g.,cholesterol, ceramides etc.). Methods for active encapsulation of matterin such synthetic liposomes, which are mainly based on membrane pHgradient or active transporters, are also well known in the art and maybe used to create synthetic liposomes with high encapsulationefficiency.

Fusion between CDLs and other liposomes to create “Hybrid CDLs” can bereadily and easily accomplished by adding short chain free PEG (˜200-500Da) to the liposomes. The mechanism of PEG-induced vesicle fusion isbelieved to be related to the reduction of water activity and thedehydration of the lipid headgroups which consequently leads to vesiclecoagulation and fusion. Fusion can also be artificially induced throughelectroporation in a process known as electrofusion. It is believed thatthis phenomenon results from the energetically active edges formedduring electroporation, which can act as the local defect point tonucleate stalk growth between two bilayers. Fusion can also be achievedby addition of detergents (usually under 2%) to the liposomal mixture(e.g., Cymal-5™, 1-S-Octyl Beta-D-thioglucopyranoside etc.), incubationwith mild agitation and consequent detergent dialysis.

Example 6 Proteomics Analysis of hMSCc Ghost and Derived CDLs

Method

Proteomics analysis was conducted on 4 samples containing ghost cellsand CDLs derived from hMSCc that were either conditioned orunconditioned by a medium derived from a prostate cancer cell-line(PC3). For the production of conditioned ghosts and CDLs, hMSCs wereincubated for 24 prior to harvesting in medium composed of 50%conditioning media derived from PC3 cells. Cells were then harvested andsonicated ghosts and CDLs were prepared thereof by the method previouslydescribed. Sonicated ghosts from conditioned and unconditioned hMSCs(10⁶ cells) were resuspended for analysis in 1 ml TM-buffer, pH 7.4.Cell-derived liposomes derived from 7×10⁶ conditioned and unconditionedhMSCc were resuspended in 50 μL TM buffer, pH 8.6.

The samples were sent for proteomics analysis at the proteomics centerof the TECHNION—Israel Institute of Technology. Briefly, the sampleswere digested by Trypsin and the resulting peptides were analyzed byLC-MS/MS. Peptide mix was fractionated by HPLC and electro-sprayed ontoan ion-trap mass spectrometer (Orbitrap™). Mass spectrometry wasperformed in order to analyze the peptides' mass to charge ratio spectraand to determine the proteins' mass. For additional analysis andidentification, the peptides were further fragmented by collisioninduced dissociation (CID) and analyzed again. The peptides wereidentified by Sequest 3.31 software against the human part of theuniprot database. All protein results are given as Uniport AccessionNumbers. The following values were determined for each protein/accessionnumber:

MW—Molecular weightP_(pro)—The probability of finding a match as good as or better than theobserved match by chance. The value displayed for the protein is theprobability of the best peptide match (the peptide with the lowestscore).Pep Count—The total number of identified peptides.Mean—The Average of the peak areas of top 3 identified peptides perprotein.Mean.SE—Mean standard error.Med—Median of the peak area of all identified peptides per proteinMedErr—Median absolute deviation.

Protein Name.

Results

The hundreds of proteins that were identified on one or more of the foursamples can be divided into 4 distinct groups:

-   -   1. Proteins that were prevalent in all four samples (Table 7),        i.e. conditioned and unconditioned ghosts and CDLs.    -   2. Proteins that were prevalent in the ghosts or conditioned        ghosts but were missing from the CDLs (Table 5). These proteins        are probably or mostly the remains of cytoplasmatic matter that        was not completely removed from the ghosts.    -   3. Proteins that were prevalent only on the conditioned ghosts        and CDLs (Table 6). These proteins are probably or mostly        membarnal proteins which are only expressed after induction or        exposure to condition media.    -   4. Proteins that were prevalent in all samples but CDLs that        were produced from unconditioned hMSCs (Table 7). These proteins        are probably or mostly membarnal proteins which are expressed in        lower levels on unconditioned hMSCs and which are completely        depleted on their derived CDLs. Inducing the hMSCs with        condition media probably elevates these proteins level to the        extent they become more apparent on the conditioned CDLs.

TABLE 4 Proteins prevalent on conditioned and unconditioned ghosts andCDLs. Fold expression on conditioned Uniport CDLs relative to Accessionunconditioned Number MW CDLS Protein name P04179 24706.6 30 MoesinP62899 14453.9 26 Myosin regulatory light chain 12A P09622 54143.1 26Sodium/potassium-transporting ATPase subunit alpha-1 P02545 74094.8 2440S ribosomal protein S28 P05023 112824.1 14 Integrin beta-1 P1114270854.4 14 Cathepsin D P62701 29579.1 12 Sulfide:quinone oxidoreductaseP07602 58073.9 12 Histone H4 P21589 63327.6 11 Major vault proteinP27797 48111.9 10 Keratin O75396 24724.8 10 Elongation factor 2 P1367461011.1 10 Erlin-2 P24752 45170.7 9 60S ribosomal protein L4 P3004422012.5 9 60S ribosomal protein L18a P51659 79636.4 9 40S ribosomalprotein S21 P61604 10924.9 8 Actin P17301 129213.8 8 Acetyl-CoAacetyltransferase P38117 27826.2 8 Leucine-rich PPR motif-containingprotein P10809 61016.5 8 Ras-related protein Rab-7a P14314 59387.9 7Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2P62736 41981.8 7 60S ribosomal protein L14 Q96D15 37470 7Voltage-dependent anion-selective channel protein 3 P62241 24190.2 7Annexin A4 Q9Y2Q3 25480.3 7 Dolichyl-diphosphooligosaccharide--proteinglycosyltransferase subunit 1 P02786 84818 7 Pyruvate kinase isozymesM1/M2 P38646 73634.8 7 40S ribosomal protein S19 P62829 14856.1 7Annexin A2 Q9H4B7 50294.6 7 Reticulocalbin-3 P40926 35480.7 7Peroxiredoxin-1 P08648 114464.9 7 Malate dehydrogenase P07237 57080.8 7Prenylcysteine oxidase 1 Q70UQ0 39285 7 40S ribosomal protein S4 Q9NZM1234558.8 7 Proactivator polypeptide Q07020 21621.1 7 Heat shock cognate71 kDa protein P30040 28975.2 6 Integrin alpha-V Q09666 628705.2 6Annexin A11 Q32P28 83341.2 6 60S ribosomal protein L7a Q15155 134267.4 6Serine hydroxymethyltransferase P04040 59718.9 6 Tubulin alpha-1B chainP30048 27675.2 6 60S ribosomal protein L26-like 1 P68104 50109.2 6Integrin alpha-2 Q00325 40068.8 6 Myoferlin P30443 40820.2 6Trifunctional enzyme subunit beta P06756 115964.5 6Thioredoxin-dependent peroxide reductase Q15149 531465.9 6 ATP synthasesubunit O P16615 114682.7 6 Elongation factor Tu P14625 92411.2 6 60Sribosomal protein L11 P19105 19781.5 6 60S acidic ribosomal proteinP0-like P34897 55957.8 6 40S ribosomal protein S3 P45880 31546.5 6Signal recognition particle receptor subunit beta O15118 142073.5 6Serpin H1 P62805 11360.4 6 Isocitrate dehydrogenase [NADP] Q9953641893.5 6 Endoplasmin P36957 48698.6 6 Tubulin beta chain P02751262439.5 5 CD44 antigen P13639 95277.1 5 60S ribosomal protein L30P49411 49510.2 5 60S ribosomal protein L12 Q00610 191491.7 5 Plectin-1P55072 89265.9 5 Aldehyde dehydrogenase X P21281 56465 5 ATP synthasesubunit alpha Q16698 36044.8 5 Collagen alpha-1(I) chain P26038 67777.95 40S ribosomal protein S8 P14854 10185.7 5 ATP synthase subunit dP50454 46411.3 5 Erlin-1 P08670 53619.2 5 Vesicle-trafficking proteinSEC22b Q13423 113822.9 5 Procollagen-lysine P62847 15413.4 5 Synapticvesicle membrane protein VAT-1 homolog P00505 47445.3 5 Proteindisulfide-isomerase A6 P05556 88357 5 Niemann-Pick C1 protein P3083757202.3 5 Annexin A1 P13667 72887.1 5 Cytochrome c oxidase subunit 6B1P06733 47139.4 5 Voltage-dependent anion-selective channel protein 2P68363 50119.6 5 10 kDa heat shock protein Q15084 48091.3 5 Hemeoxygenase 1 P04844 69241.1 5 Lysosome membrane protein 2 P36578 47667.55 60S ribosomal protein L22 P11021 72288.5 5 Dipeptidyl peptidase 4O60568 84731.7 5 ATP synthase subunit beta P04406 36030.4 5 KeratinO95816 23757.2 5 Calreticulin Q04837 17249 5 60S acidic ribosomalprotein P2 P09601 32798 5 Transitional endoplasmic reticulum ATPaseQ06830 22096.3 5 Lamin-A/C Q9H9B4 35596.4 5 Protein disulfide-isomeraseA3 P09525 35860.1 5 40S ribosomal protein S5 P16070 81503.4 5 Tubulinbeta-1 chain P04075 39395.3 5 Annexin A5 P14649 22749.7 5 Electrontransfer flavoprotein subunit beta Q02809 83497.5 5 Peptidyl-prolylcis-trans isomerase B P10606 13686.9 5 Collagen alpha-1(VI) chain P4873550876.9 5 Translocon-associated protein subunit delta P07339 44523.7 5Keratin Q9Y6N5 49928.9 5 Peroxisomal multifunctional enzyme type 2Q7KZF4 101933.6 5 Prohibitin-2 P50213 39566.1 5 Leucine-richrepeat-containing protein 59 Q9UNX3 17245.6 5 Prolyl 3-hydroxylase 1P14618 57900.2 5 Lysosome-associated membrane glycoprotein 2 Q96AG434908.9 5 40S ribosomal protein S18 P07355 38579.8 4 Guaninenucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 P61803 12488.64 Dihydrolipoyl dehydrogenase Q13162 30520.8 4 60S ribosomal protein L5P63220 9105.6 4 Guanine nucleotide-binding protein subunit beta-2-like 1P62263 16262.5 4 60S ribosomal protein L18 Q14108 54255.6 4 Histone H2Btype 1-B P63244 35054.6 4 5′-nucleotidase P49748 70345.5 4 ADP/ATPtranslocase 2 P55084 51261.6 4 Aminopeptidase N P42704 157804.6 4L-lactate dehydrogenase A chain P00338 36665.4 4 Voltage-dependentanion-selective channel protein 1 Q02543 20748.9 4 Glucosidase 2 subunitbeta Q13885 49875 4 Annexin A6 P30101 56746.8 4 ATP synthase subunitdelta P35268 14777.8 4 Peroxiredoxin-5 Q9P2E9 152380 4 Very long-chainspecific acyl-CoA dehydrogenase P62424 29977 4 Prolow-densitylipoprotein receptor-related protein 1 P36543 26128.8 4 Inhibitor ofnuclear factor kappa-B kinase-interacting protein P62857 7836.2 4 40Sribosomal protein S24 P62753 28663 4 Elongation factor 1-alpha 1 Q9UHG356603.8 4 Aspartate aminotransferase P50914 23417 4 60S ribosomalprotein L13 P60174 26652.7 4 Adipocyte plasma membrane-associatedprotein P27487 88222.5 4 Fibronectin P62269 17707.9 4 Myosin light chain6B P36542 32975.3 4 Transgelin P39656 50769 4 Staphylococcal nucleasedomain-containing protein 1 Q99623 33275.9 4 Sideroflexin-3 P2637324246.5 4 60S ribosomal protein L31 Q9HDC9 46450.9 4 Phosphate carrierprotein Q71U36 50103.7 4 ADP/ATP translocase 1 P08865 32833.4 4Lysosome-associated membrane glycoprotein 1 P00387 34212.7 4Hexokinase-1 Q9Y5M8 29683.8 4 Protein disulfide-isomerase A4 P2570559713.7 4 Catalase P23284 23727.5 4 Cytochrome c oxidase subunit 5AP05387 11657.9 4 40S ribosomal protein SA P43307 32215.4 4 Isocitratedehydrogenase [NAD] subunit alpha P49755 24960 4 2 P04083 38690 4Tubulin beta-2C chain P04843 68526.9 4 V-type proton ATPase subunit E 1P08133 75825.7 4 40S ribosomal protein S6 P68371 49799 4 V-type protonATPase catalytic subunit A O75477 38901.4 4 Trifunctional enzyme subunitalpha P21796 30753.6 4 Ribosome-binding protein 1 P62873 37353 4Vimentin P12235 33043.2 4 Protein S100-A11 P20674 16751.7 4Procollagen-lysine Q9BWM7 35480.5 4 Mitochondrial inner membrane proteinP23396 26671.4 4 60S ribosomal protein L23 P05141 32874.2 4 60 kDa heatshock protein P61247 29925.8 3 Alpha-enolase Q16891 83626.5 3Transmembrane emp24 domain-containing protein 9 P51571 18986.6 3Collagen alpha-2(I) chain O75489 30222.7 3 Endoplasmic reticulumresident protein 29 P31930 52612.5 3 Cytochrome b-c1 complex subunit 1P48047 23262.7 3 NADH dehydrogenase [ubiquinone] iron-sulfur protein 3P62888 12775.7 3 Neuroblast differentiation-associated protein AHNAKP12111 343450.3 3 Prohibitin Q8NHW5 34342.7 3 40S ribosomal protein S14P12109 108462 3 V-type proton ATPase subunit B P30050 17807.5 3Triosephosphate isomerase P40939 82947 3 Integrin alpha-5 O75947 18479.53 Fructose-bisphosphate aldolase A P31040 72645.4 3 Calnexin P19367102420.2 3 Cytochrome b-c1 complex subunit 2 P30049 17479.2 3 Prolyl4-hydroxylase subunit alpha-1 P46782 22862.1 3 Transmembrane emp24domain-containing protein 10 P39019 16050.5 3 Peroxiredoxin-4 Q0706565982.9 3 Stomatin-like protein 2 P35232 29785.9 3 BAG family molecularchaperone regulator 2 P62913 20239.7 3 Sarcoplasmic/endoplasmicreticulum calcium ATPase 2 P06576 56524.7 3 Clathrin heavy chain 1P51149 23474.9 3 Collagen alpha-3(VI) chain P27824 67526 3 60S ribosomalprotein L9 P31949 11732.8 3 HLA class I histocompatibility antigenP35579 226390.6 3 NADH-ubiquinone oxidoreductase 75 kDa subunit P02452138826.8 3 Dolichyl-diphosphooligosaccharide--proteinglycosyltransferase 48 kDa subunit Q01995 22596.4 3Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunitDAD1 Q14764 99266.1 3 Transferrin receptor protein 1 Q9UJZ1 38510.2 340S ribosomal protein S3a P08123 129209.8 3 NADH-cytochrome b5 reductase3 P50995 54355.1 2 Sideroflexin-1 P11279 44853.9 2 Actin Q9BVK6 27260.22 Myosin-9 P38606 68260.6 2 NAD(P) transhydrogenase P08758 35914.4 2 60Sribosomal protein L7 P28331 79416.7 2 CD59 glycoprotein P15144 109470.82 Cytochrome c oxidase subunit 5B P22695 48412.9 2Glyceraldehyde-3-phosphate dehydrogenase P35908 65393.2 2 GlutathioneS-transferase kappa 1 P35527 62026.7 2 Tubulin alpha-1A chain Q07954504243.2 2 Dihydrolipoyllysine-residue succinyltransferase component of2-oxoglutarate dehydrogenase complex P33778 13941.6 2 Succinatedehydrogenase [ubiquinone] flavoprotein subunit P13645 58791.6 1Cytoskeleton-associated protein 4 P60709 41709.7 1 Keratin P4677734340.7 1 Stress-70 protein Q9Y277 30639.3 1 Protein disulfide-isomeraseP32969 21849.8 1 ATP synthase subunit gamma P18124 29207.2 1Translocon-associated protein subunit alpha O94905 37815.5 1Single-stranded DNA-binding protein P13987 14167.8 1 78 kDaglucose-regulated protein P07437 49639 1 Nodal modulator 1 P1347344932.3 1 Superoxide dismutase [Mn] P04264 65998.9 1 Tubulin beta-2Achain

TABLE 5 Proteins that were prevalent in the ghosts or conditioned ghostsbut were missing from the CDLs Uniport Prevalent in Accession Prevalentconditioned Number MW in ghosts ghosts Protein name P78527 468786.9 + +Actin-related protein 2/3 complex subunit 1B Q99715 332939.7 + + rRNA2′-O-methyltransferase fibrillarin Q14573 303910.4 + + NADHdehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8 P24821240697.7 + + E3 ubiquitin-protein ligase MARCH5 P35580 228856.9 + + Acylcarrier protein P07942 197935.7 + + Fibrillin-1 P55268 195853.3 + +Extracellular sulfatase Sulf-1 Q8WWI1 192589.5 + + Protein kinase Calpha type P20908 183446.3 + + Cytosol aminopeptidase P11047177487.9 + + Beta-galactosidase Q6YHK3 161586.8 + + Histone H3.1t Q7L576145088.6 + + GDP-fucose protein O-fucosyltransferase 1 Q08211140868.9 + + Protein S100-A6 P23634 137832.9 + + Protein tyrosinephosphatase-like protein PTPLAD1 Q12768 134200.9 + + 39S ribosomalprotein L43 P00533 134190.2 + + Radixin O43795 131901.9 + + Putativeribosomal RNA methyltransferase NOP2 Q9BSJ8 122780.1 + + Trophoblastglycoprotein P26006 118680.2 + + Galectin-3 P54707 115437.2 + + ATPsynthase subunit f Q8N766 111689.2 + + Calponin-2 O60313 111560.7 + +Tumor necrosis factor receptor superfamily member 10B Q9Y4L1111266.2 + + Probable glutathione peroxidase 8 Q6P179 110391.1 + +Hydroxyacyl-coenzyme A dehydrogenase P22413 104856.7 + + Medium-chainspecific acyl-CoA dehydrogenase Q6ZXV5 103941.9 + + SH3 domain-bindingglutamic acid-rich-like protein 3 A0FGR8 102294.1 + + V-type protonATPase 116 kDa subunit a isoform 3 P11586 101495.6 + + U5 small nuclearribonucleoprotein 200 kDa helicase Q15063 93255.4 + + Transducinbeta-like protein 2 Q13488 92908.6 + + Extended synaptotagmin-2 O9547988836.6 + + Mitochondrial import inner membrane translocase subunit Tim8A Q9UBV2 88698.6 + + Histone H1.2 Q9NR30 87290.5 + + Vesicularintegral-membrane protein VIP36 Q15436 86105.3 + + SRAstem-loop-interacting RNA-binding protein Q99798 85372 + + Nuclear porecomplex protein Nup205 P08238 83212.2 + + DnaJ homolog subfamily Bmember 1 P13010 82652.4 + + HEAT repeat-containing protein 1 Q96TA182631.1 + + Pyruvate dehydrogenase E1 component subunit alpha Q8IVL681785.8 + + Pre-mRNA-processing-splicing factor 8 Q9UH99 80261.7 + +Collagen alpha-1(XIV) chain Q9BU23 79647.6 + + Probable saccharopinedehydrogenase Q96AC1 77810.7 + + Nucleoside diphosphate kinase B P2198077279.8 + + Protein DEK Q6NUQ4 77101.6 + + Nascentpolypeptide-associated complex subunit alpha P17252 76714.3 + + LIMdomain only protein 7 P23246 76101.8 + + NADH dehydrogenase [ubiquinone]iron-sulfur protein 6 Q99805 75725.7 + + Peptidyl-tRNA hydrolase 2O75746 74715 + + Deoxyribonuclease-2-alpha P46063 73410 + +Alpha-L-iduronidase Q8NBJ5 71590.6 + + Cytochrome c oxidase subunit 6CP17066 70984.4 + + Signal peptidase complex subunit 2 O43390 70899.2 + +60S ribosomal protein L35 P43155 70812.5 + + Adenosine 3′-phospho5′-phosphosulfate transporter 1 P34931 70331.5 + +Proliferation-associated protein 2G4 P54652 69978 + + Splicing factorP17844 69104.8 + + Ras-related protein Ral-A Q96CM8 68080.8 + + 60Sribosomal protein L10-like Q03252 67647.6 + + ATP-binding cassettesub-family E member 1 O94826 67412.2 + + Actin-related protein 2/3complex subunit 2 P20700 66367.7 + + Cathepsin Z Q5JTV8 66208.4 + +Acyl-coenzyme A thioesterase 1 O00567 66008.8 + + Signal peptidasecomplex catalytic subunit SEC11A P23368 65402 + + Acetyl-coenzyme Atransporter 1 Q10471 64691.5 + + 4F2 cell-surface antigen heavy chainQ10472 64177.5 + + Tropomyosin beta chain P14866 64092.4 + + Coiled-coildomain-containing protein 47 Q14956 63882 + + Myb-binding protein 1AP07686 63071.3 + + ADP-ribosylation factor 1 O95302 63043.6 + +Synaptonemal complex protein SC65 Q969V3 62934.7 + + Signal peptidasecomplex subunit 3 P30038 61680.7 + + NADH dehydrogenase [ubiquinone]iron-sulfur protein 2 Q96S52 61617.3 + + Ras-related protein Rab-8AQ9HCC0 61294.5 + + EH domain-containing protein 2 Q5SSJ5 61169.3 + + RhoGTPase-activating protein 1 Q9P0J1 61015.7 + + Putative 40S ribosomalprotein S26-like 1 Q9H857 60679.8 + + Mitochondrial chaperone BCS1P04062 59678.4 + + Calcium-binding mitochondrial carrier protein Aralar2Q9Y2X3 59540.6 + + Ribosome-releasing factor 2 Q7Z4H8 58535.3 + + 39Sribosomal protein L46 Q13217 57544.3 + + Cytochrome c1 P4925757513.1 + + Transmembrane 9 superfamily member 4 P26599 57185.8 + + 60Sribosomal protein L36a P05091 56345.7 + + Metaxin-2 Q96A33 55838.4 + +Heterogeneous nuclear ribonucleoprotein L Q9UMS4 55146.4 + + ES1 proteinhomolog O60701 54989.3 + + Replication protein A 14 kDa subunit O7602154939 + + 60S ribosomal protein L37a P10619 54431.2 + + Neuron-specificcalcium-binding protein hippocalcin Q96HE7 54358.1 + + Translocationprotein SEC63 homolog Q15233 54197.4 + + Protein transport protein Sec61subunit beta Q02818 53846.4 + + Torsin-1A-interacting protein 2 P2257053803.1 + + NADH dehydrogenase [ubiquinone] iron-sulfur protein 7 Q96N6652730.3 + + Fatty aldehyde dehydrogenase P20073 52705.8 + +All-trans-retinol 13 P61619 52230.6 + + H/ACA ribonucleoprotein complexsubunit 4 Q9Y512 51943.4 + + Multidrug resistance-associated protein 1O43615 51323.6 + + Neuroplastin Q07960 50404.3 + + Heterochromatinprotein 1-binding protein 3 Q13509 50400.3 + + Transmembrane emp24domain-containing protein 3 Q9P2R7 50285.3 + + Phosphoglycerate mutase 1P80303 50164.4 + + Putative heterogeneous nuclear ribonucleoproteinA1-like 3 P36551 50120.1 + + Isovaleryl-CoA dehydrogenase P1348949941.2 + + ATP synthase subunit b Q9Y305 49869.6 + + Ras-relatedprotein Rab-5A Q9BUF5 49825 + + Poly(rC)-binding protein 2 P0435049553.9 + + Actin-related protein 2/3 complex subunit 5 P3194349198.4 + + 60S ribosomal protein L13a P62495 49000.2 + + Mitochondrialimport inner membrane translocase subunit Tim13 P82675 47976.2 + +Beta-actin-like protein 2 P09543 47548.7 + + Protein disulfide-isomeraseTMX3 P45954 47455.3 + + Acid ceramidase Q8NBX0 47121.5 + + Lipasematuration factor 2 O60664 47018 + + Ras-related C3 botulinum toxinsubstrate 1 P28300 46914.5 + + Golgin subfamily B member 1 P1131046558.6 + + Ectonucleotide pyrophosphatase/phosphodiesterase familymember 1 O75718 46532 + + Ras-related protein Rab-18 O14979 46409 + +High mobility group protein B1 P26440 46289.7 + + Coproporphyrinogen-IIIoxidase P60842 46124.6 + + Contactin-associated protein 1 Q58FF345829.9 + + Protein S100-A16 Q96HD1 45408.9 + + Isocitrate dehydrogenase[NAD] subunit beta Q6YN16 45365.5 + + CDP-diacylglycerol--inositol3-phosphatidyltransferase Q8NC51 44938.5 + + Follistatin-related protein1 Q96G23 44847.4 + + Ribose-phosphate pyrophosphokinase 1 Q9BTV444847.3 + + Eukaryotic initiation factor 4A-III P61160 44732.3 + +Probable cation-transporting ATPase 13A1 P09110 44263.9 + + Highmobility group protein HMGI-C P07093 43974.3 + + Mesoderm-specifictranscript homolog protein Q9H488 43927.2 + + CytoplasmicFMR1-interacting protein 1 O75521 43557.4 + + Matrin-3 Q6NVY143454.4 + + Polypeptide N-acetylgalactosaminyltransferase 1 P1730242980.9 + + 39S ribosomal protein L1 Q13336 42499.8 + +Dolichol-phosphate mannosyltransferase Q16795 42482.6 + +Peptidyl-prolyl cis-trans isomerase C P35613 42174.1 + + Gamma-glutamylhydrolase P29992 42096.6 + + V-type proton ATPase 16 kDa proteolipidsubunit Q9BYX7 41988.9 + + Ribosome biogenesis regulatory proteinhomolog Q562R1 41976 + + 28S ribosomal protein S5 Q9BRK5 41780.5 + +Lanosterol synthase P30533 41440.9 + + Glyoxylatereductase/hydroxypyruvate reductase P82650 41254.4 + + Transforminggrowth factor-beta-induced protein ig-h3 Q15050 41168.2 + +Beta-actin-like protein 3 Q12907 40203.1 + + Nucleolar RNA helicase 2O75367 39592.5 + + CAAX prenyl protease 1 homolog Q9NYL9 39570.3 + +RNA-binding protein FUS P09972 39431.3 + + ADP-ribosylation factor-likeprotein 6-interacting protein 1 Q5EB52 38805.5 + + Glia-derived nexinQ15366 38555.6 + + Tubulin beta-6 chain Q9H0U3 38011.4 + + Magnesiumtransporter protein 1 O15121 37841.1 + + 60S ribosomal protein L6 Q9UDY437783.2 + + Peptidyl-prolyl cis-trans isomerase FKBP3 P62136 37487.8 + +Up-regulated during skeletal muscle growth protein 5 Q14257 36853.7 + +Thioredoxin-related transmembrane protein 1 P05198 36089.4 + + 28Sribosomal protein S31 Q9NZ01 36010.8 + + High mobility group proteinHMG-I/HMG-Y P27695 35532.2 + + Antigen peptide transporter 1 P0857435367 + + DnaJ homolog subfamily C member 3 P31937 35305.8 + +Peroxisomal acyl-coenzyme A oxidase 1 Q08257 35184.6 + + Chlorideintracellular channel protein 1 Q15006 34811.4 + +N-acetylglucosamine-6-sulfatase P09486 34609.7 + + Probabletranscription factor PML O15144 34311.5 + + Mitochondrial importreceptor subunit TOM70 Q16836 34255.9 + + Endoplasmic reticulumaminopeptidase 2 Q9UHQ9 34073.2 + + LDLR chaperone MESD P53007 33991 + +Acyl-coenzyme A thioesterase 13 Q9UBR2 33846.2 + + Lamin-B1 Q8NBJ733835.8 + + 116 kDa U5 small nuclear ribonucleoprotein component P6299533645.6 + + Proteolipid protein 2 Q9BPW8 33288.9 + + Collagen triplehelix repeat-containing protein 1 P07951 32830.6 + + Isocitratedehydrogenase [NAD] subunit gamma P42126 32795.2 + + RNA-bindingRaly-like protein Q02878 32707.6 + + Sphingolipid delta(4)-desaturaseDES1 Q86SE5 32310.6 + + 3 Q9Y639 31271.9 + + Mitochondrial import innermembrane translocase subunit TIM44 P15559 30848 + + Mammalianependymin-related protein 1 O75431 29744.1 + + Aldehyde dehydrogenaseO60762 29615.8 + + Urea transporter 1 P22090 29437 + + Retinoldehydrogenase 11 P62258 29155.4 + + T-complex protein 1 subunit deltaP24539 28890.3 + + Ribonuclease inhibitor P18669 28785.9 + + Splicingfactor P67936 28504.5 + + 60S ribosomal protein L28 Q9UFN0 28448.5 + +Calpain small subunit 1 Q9UHQ4 28302.2 + + Histone H1.1 P3004228152.7 + + Pre-mRNA-processing factor 19 P57088 27959.8 + + NADHdehydrogenase [ubiquinone] iron-sulfur protein 5 Q9P0L0 27875.2 + + OCIAdomain-containing protein 2 Q07955 27727.8 + + Histone H1.0 P6310427727.7 + + Glycylpeptide N-tetradecanoyltransferase 1 Q9NR2827113.7 + + Putative 60S ribosomal protein L13a-like MGC87657 P3331626689.7 + + Major facilitator superfamily domain-containing protein 10O75352 26620.5 + + Transmembrane emp24 domain-containing protein 1P54819 26460.8 + + Cystatin-B P17931 26136.1 + + Integrin alpha-3 P6003325792.1 + + Signal peptidase complex subunit 1 Q9UM22 25420.6 + +NAD(P)H dehydrogenase [quinone] 1 Q13445 25189.7 + + Mannose-P-dolicholutilization defect 1 protein Q00688 25161.3 + + DnaJ homolog subfamily Bmember 4 Q15005 24986.7 + + Heat shock 70 kDa protein 6 P0942924878.2 + + Heterogeneous nuclear ribonucleoprotein D-like P6290624815.5 + + Protein Mpv17 Q9Y3Q3 24761.3 + + Tubulin beta-3 chain P2763524587.9 + + Anoctamin-10 Q96L21 24502.7 + + Acyl-CoA synthetase familymember 2 P62826 24407.6 + + Heat shock protein beta-1 B2RPK0 24222.8 + +Peptidyl-prolyl cis-trans isomerase FKBP7 O43402 23757.7 + + Thioredoxinreductase 2 P20339 23643.8 + + Acyl-coenzyme A thioesterase 9 P4042923562.4 + + Heterogeneous nuclear ribonucleoprotein H P11233 23552 + +Probable ATP-dependent RNA helicase DDX5 O14735 23523.1 + +Hydroxysteroid dehydrogenase-like protein 2 Q8N983 23416.2 + + WASHcomplex subunit strumpellin P45877 22748.8 + + NADH dehydrogenase[ubiquinone] 1 alpha subcomplex subunit 9 P46781 22577.6 + + Nucleosomeassembly protein 1-like 1 P84074 22413 + + Lysosomal protective proteinQ96AB3 22322.8 + + Beta-hexosaminidase subunit alpha O43399 22224.3 + +Serum albumin Q02539 21828.9 + + B-cell receptor-associated protein 29O75915 21600.4 + + Mitochondrial import inner membrane translocasesubunit TIM50 Q9H061 21513.5 + + NADH dehydrogenase [ubiquinone]flavoprotein 2 P16403 21351.8 + + Protein sel-1 homolog 1 P8407720683.7 + + Beta-hexosaminidase subunit beta P67812 20612.1 + +Nucleolar protein 56 P18085 20497.7 + + Sterol-4-alpha-carboxylate3-dehydrogenase P24844 19814.5 + + WASH complex subunit 7 O6083119245.5 + + Glycine cleavage system H protein P46778 18553.1 + +Putative hexokinase HKDC1 P62280 18419 + + SWI/SNF complex subunitSMARCC2 P62277 17211.7 + + 39S ribosomal protein L38 Q9BX68 17151.2 + +Ganglioside GM2 activator P15531 17137.7 + + Oligosaccharyltransferasecomplex subunit OSTC P62841 17029.2 + + Small nuclearribonucleoprotein-associated proteins B and B′ P63241 16821.4 + +Regulator of chromosome condensation Q9NRP0 16817.8 + + Nucleosidediphosphate kinase A Q86SX6 16617.5 + + 60S ribosomal protein L34 O1551116310.3 + + Tubulin beta-4 chain P46779 15737.7 + + Tropomyosin alpha-4chain P26885 15639.3 + + Receptor expression-enhancing protein 5 O6036115519 + + Putative pre-mRNA-splicing factor ATP-dependent RNA helicaseDHX15 Q9NS69 15511.8 + + Four and a half LIM domains protein 2 Q9Y3E015415.4 + + ATP-dependent RNA helicase DHX29 P69905 15247.9 + +Leucyl-cystinyl aminopeptidase P42766 14542.6 + + Heterogeneous nuclearribonucleoprotein R P55769 14164.6 + + Histidyl-tRNA synthetase P0490814127 + + Small nuclear ribonucleoprotein Sm D3 P61769 13705.9 + +Prostacyclin synthase Q5JNZ5 12994 + + [Pyruvate dehydrogenase[acetyl-transferring]]- phosphatase 1 Q9GZT3 12341.4 + + Proteintransport protein Sec23A P84090 12251 + + Abhydrolase domain-containingprotein 10 Q96FQ6 11794 + + Putative endoplasmin-like protein P9999911741.1 + + 60S acidic ribosomal protein P1 P17096 11669.2 + + Trans-2O60220 10991.3 + + GDH/6PGL endoplasmic bifunctional protein P5613410910.7 + + Potassium-transporting ATPase alpha chain 2 Q9Y5L4 10493 + +Eukaryotic peptide chain release factor subunit 1 Q9H299 10431.3 + +Transmembrane and TPR repeat-containing protein 3 P61513 10268.5 + +Ribosomal L1 domain-containing protein 1 O75531 10052 + + Regulator ofmicrotubule dynamics protein 1 P60468 9968.1 + + Adenylate kinase 2P62979 9411.9 + + Alpha-2-macroglobulin P63173 8212.7 + + 72 kDa type IVcollagenase P04732 6009.2 + + SUN domain-containing protein 1 P35555312082 + − Laminin subunit beta-1 Q05707 193393 + − SUNdomain-containing protein 2 Q7Z478 155138.3 + − Vesicle transportprotein GOT1B Q9HD20 132869.9 + − Actin-related protein 2 Q9UIQ6117274.2 + − Hemoglobin subunit alpha Q9UGP8 87941.5 + − ERO1-likeprotein alpha Q9UJS0 74128.8 + − Glucosylceramidase P27658 73317.2 + −Metaxin-1 P35475 72624.8 + − ATP-dependent DNA helicase Q1 P0276869321.6 + − Tumor protein D54 P35241 68521.5 + − Epidermal growth factorreceptor P08195 67951.9 + − PolypeptideN-acetylgalactosaminyltransferase 2 Q6NUM9 66776.8 + − Annexin A7 Q9NNW766320.8 + − Neighbor of COX4 Q86UE4 63798.8 + − Actin-related protein2/3 complex subunit 3 P12081 57374.3 + − NHP2-like protein 1 Q9UBM754453.8 + − Cleft lip and palate transmembrane protein 1 P4918953767.1 + − ATP-binding cassette sub-family D member 3 O15269 52710.6 +− Arylsulfatase A O75306 52511.8 + − Delta-1-pyrroline-5-carboxylatedehydrogenase P12694 50439.2 + − ADP-dependent glucokinase Q9279150349.3 + − Peptidyl-prolyl cis-trans isomerase FKBP9 Q15113 47942 + −Prostaglandin E synthase 2 Q8TB61 47483.6 + − CarnitineO-acetyltransferase P55209 45346 + − 40S ribosomal protein S9 Q9266545290.5 + − Eukaryotic translation initiation factor 2 subunit 1 Q96DV444568.4 + − 40S ribosomal protein S13 Q13561 44203.9 + − Coiled-coildomain-containing protein 109A Q9UQ80 43759.2 + − Heat shock 70 kDaprotein 1-like P29803 42905.6 + − Niban-like protein 1 P51553 42767.1 +− 3-hydroxyisobutyryl-CoA hydrolase P61163 42586.9 + − Splicing factorP25685 38020.4 + − Heat shock protein HSP 90-beta Q9H2U2 37896 + − CD166antigen Q9H7B2 35560.2 + − Transient receptor potential cation channelsubfamily V member 2 Q8TC12 35363.5 + − 40S ribosomal protein S4 Q1284134962.9 + − Plasminogen activator inhibitor 1 RNA-binding protein P6089134811.9 + − LAG1 longevity assurance homolog 2 Q99439 33675.3 + −Uncharacterized protein KIAA0090 Q86WA6 32522 + − Putative phospholipaseB-like 2 Q14192 32170.6 + − Mitochondrial import receptor subunit TOM22homolog P16152 30355.9 + − 28S ribosomal protein S23 Q9Y680 29990.1 + −Putative high mobility group protein B1-like 1 O60613 27629 + −ATP-dependent RNA helicase DDX18 Q96CG8 26207.1 + − Protein NipSnaphomolog 1 Q14696 26060.3 + − NADH-cytochrome b5 reductase 1 Q1376523369.7 + − Transmembrane protein 214 P09211 23341 + − 40S ribosomalprotein S29 P04792 22768.5 + − GTP-binding nuclear protein Ran Q9Y3D921757.3 + − Carbonyl reductase [NADPH] 1 Q00765 21479.1 + −Peptidyl-prolyl cis-trans isomerase FKBP2 P63000 21436.3 + − Perilipin-3P05976 21131.8 + − Nuclear pore complex protein Nup155 P30086 21043.7 +− Vesicle-associated membrane protein 2 Q9ULC4 20541.8 + − Succinyl-CoAligase [ADP-forming] subunit beta P61009 20300.5 + − Nicalin P5197020092.1 + − Inositol 1 P84103 19317.9 + − Malignant T cell-amplifiedsequence 1 Q9Y6H1 15502.7 + − U1 small nuclear ribonucleoprotein CQ9NPJ3 14950.9 + − Tricarboxylate transport protein P35244 13559.9 + −UDP-glucose 6-dehydrogenase O43920 12509.4 + − Transmembrane protein 33Q6NVV1 12126.9 + − Diablo homolog P04080 11132.6 + − Non-POUdomain-containing octamer-binding protein O43678 10914.8 + − AnkycorbinP09669 8775.7 + − Procollagen galactosyltransferase 1 Q15738 NA − +ADP-ribosylation factor 4 P17900 NA − + Histidine triadnucleotide-binding protein 2 P46087 NA − + Myosin-Ib O00299 NA − +Quinone oxidoreductase P39210 NA − + 60S ribosomal protein L10a Q9P035NA − + Plasma membrane calcium-transporting ATPase 4 Q9NUJ1 NA − +Enhancer of rudimentary homolog O43837 NA − + Cysteine-rich withEGF-like domain protein 1 Q9Y5S1 NA − + Ribosome production factor 2homolog Q16647 NA − + Beta-2-microglobulin Q96JJ7 NA − + 2′ P23434 NA− + PRA1 family protein 2 POC7M2 NA − + Nucleobindin-2 P15289 NA − +Serine palmitoyltransferase 1 O75844 NA − + Core histone macro-H2A.1P09234 NA − + Coiled-coil-helix-coiled-coil-helix domain-containingprotein 2 P08253 NA − + 60S ribosomal protein L38 P04632 NA − + ProteinNipSnap homolog 3A Q03518 NA − + DNA-(apurinic or apyrimidinic site)lyase Q8NFQ8 NA − + Nucleobindin-1 P07305 NA − + Splicing factor Q15029NA − + Sulfatase-modifying factor 2 O75643 NA − + C-1-tetrahydrofolatesynthase Q9Y276 NA − + 5′-nucleotidase domain-containing protein 2Q9BYD6 NA − + Gap junction alpha-1 protein Q8IWU6 NA − + Laminin subunitbeta-2 Q9UBQ7 NA − + Alpha-2-macroglobulin receptor-associated proteinP61221 NA − + Lamin-B2 P63027 NA − + Periostin Q9Y4P3 NA − +Phosphatidylethanolamine-binding protein 1 P30419 NA − + 14-3-3 proteinzeta/delta P50991 NA − + 14-3-3 protein epsilon O94901 NA − +Metallothionein-1E Q9H7Z7 NA − + Procollagen C-endopeptidase enhancer 1P22087 NA − + Collagen alpha-1(XII) chain P28288 NA − +4-trimethylaminobutyraldehyde dehydrogenase Q13505 NA − + Collagenalpha-1(VIII) chain Q9Y3E5 NA − + Transmembrane 9 superfamily member 2P27449 NA − + Guanine nucleotide-binding protein subunit alpha-11 Q13510NA − + Short/branched chain specific acyl-CoA dehydrogenase P62318 NA− + Histone H2A type 1-B/E O00115 NA − + Calcium-binding mitochondrialcarrier protein Aralar1 P33527 NA − + Sorting and assembly machinerycomponent 50 homolog Q8NE86 NA − + Dynactin subunit 2 O60832 NA − +Protein transport protein Sec61 subunit alpha isoform 1 Q15582 NA − +28S ribosomal protein S22 Q13740 NA − + Inorganic pyrophosphatase 2Q9NVP1 NA − + 15 kDa selenoprotein P22392 NA − + Fermitin family homolog2 P43243 NA − + Peroxisomal 3 P38919 NA − + Transmembrane protein 43Q04941 NA − + Transformer-2 protein homolog beta P05386 NA − +Cytochrome c Q8NHP8 NA − + 40S ribosomal protein S11 Q8TAQ2 NA − +Valacyclovir hydrolase O15145 NA − + Protein LYRIC Q86TX2 NA − +Torsin-1A-interacting protein 1 Q16629 NA − + Alpha-centractin P35637 NA− + Tropomodulin-3 Q9NX47 NA − + Cartilage-associated protein Q9NP72 NA− + Tenascin Q9NZN4 NA − + Methylcrotonoyl-CoA carboxylase beta chainP51648 NA − + Lysophospholipid acyltransferase 7 O15143 NA − +DNA-dependent protein kinase catalytic subunit Q92820 NA − + BasiginQ9Y6A9 NA − + CD81 antigen P29590 NA − + SPARC Q6P2Q9 NA − + Prolyl3-hydroxylase 3 Q13641 NA − + 60S ribosomal protein L21 Q2TB90 NA − +Extended synaptotagmin-1 Q14789 NA − + Protein-lysine 6-oxidase P15586NA − + Tetratricopeptide repeat protein 35 O75694 NA − + Myosin lightchain 1/3 Q56VL3 NA − + Vesicle-associated membrane protein-associatedprotein A P61006 NA − + GPI transamidase component PIG-S P49207 NA − +Glutaredoxin-related protein 5 P14678 NA − + 40S ribosomal protein S15Q96IX5 NA − + Serine/threonine-protein phosphatase PP1-alpha catalyticsubunit O75380 NA − + Splicing factor Q969S9 NA − + Nucleolar protein 58P52926 NA − + 3-ketoacyl-CoA thiolase Q9H2W6 NA − + KDELmotif-containing protein 2 Q08170 NA − + Heat shock-related 70 kDaprotein 2 O14763 NA − + Dynamin-like 120 kDa protein Q15067 NA − +3-hydroxyisobutyrate dehydrogenase Q2M389 NA − + Myosin regulatory lightpolypeptide 9 Q3ZCQ8 NA − + PRA1 family protein 3 Q9BRR6 NA − +2-oxoisovalerate dehydrogenase subunit alpha P78357 NA − + Eukaryoticinitiation factor 4A-I P19404 NA − + Transmembrane protein 126A P35659NA − + Protein-glutamine gamma-glutamyltransferase 2 P16278 NA − +Laminin subunit gamma-1 Q9P0K7 NA − + NADH dehydrogenase [ubiquinone] 1alpha subcomplex subunit 2 P18754 NA − + Eukaryotic translationinitiation factor 5A-1 P62273 NA − + Glutathione S-transferase P Q14728NA − + Deoxyuridine 5′-triphosphate nucleotidohydrolase O75251 NA − +Reticulocalbin-2 Q9H3N1 NA − + NADPH:adrenodoxin oxidoreductase P06703NA − + ATP-dependent RNA helicase A O96005 NA − + 7-dehydrocholesterolreductase P28838 NA − + Collagen alpha-1(V) chain P83881 NA − +Polypyrimidine tract-binding protein 1 O43143 NA − + Putative nucleosidediphosphate kinase O00400 NA − + NAD-dependent malic enzyme Q8TED1 NA− + Hypoxia up-regulated protein 1 Q9H583 NA − + X-ray repaircross-complementing protein 5 Q16695 NA − + CD109 antigen Q96DB5 NA − +Barrier-to-autointegration factor Q9NW15 NA − + 60S ribosomal proteinL10 P01023 NA − + 40S ribosomal protein S27a Q92544 NA − + ProteinERGIC-53 Q9BQG0 NA − + Transmembrane glycoprotein NMB P48449 NA − +Fructose-bisphosphate aldolase C P06865 NA − + 45 kDa calcium-bindingprotein O14561 NA − + Isochorismatase domain-containing protein 2 Q15041NA − + Aconitate hydratase Q92621 NA − + Myosin-10

TABLE 6 Proteins that were prevalent only on the conditioned ghosts andCDLs Ratio of expression: conditioned Uniport CDLs/ Accessionconditioned Number MW ghost Protein name Q15717 NA 100% Endoplasmicreticulum lectin 1 P14136 NA 100% Signal recognition particle receptorsubunit alpha Q32P51 NA 17% Long-chain-fatty-acid--CoA ligase 3 Q9NVA2NA 16% Cation-independent mannose-6- phosphate receptor O75131 NA 11%Granulins Q96DZ1 NA 10% Heterogeneous nuclear ribonucleoprotein K P01889NA 9% DnaJ homolog subfamily C member 10 Q16270 NA 9% Copine-3 P61978 NA7% Flotillin-1 P21912 NA 6% Metalloproteinase inhibitor 3 P28799 NA 6%Glial fibrillary acidic protein Q8IXB1 NA 6% Translational activatorGCN1 Q5KU26 NA 5% Heterogeneous nuclear ribonucleoprotein A1-like 2O75955 NA 4% Protein FAM98A P35625 NA 4% Septin-11 Q92616 NA 4%Succinate dehydrogenase [ubiquinone] iron-sulfur subunit O95573 NA 3%Collectin-12 Q8NCA5 NA 3% Insulin-like growth factor-binding protein 7P11717 NA 2% HLA class I histocompatibility antigen

TABLE 7 Proteins that were prevalent in ghosts, conditioned ghosts andconditioned CDLs but were missing from unconditioned CDLs. Ratio ofexpression Uniport Cond Cond. Accession Ghosts/ CDLs/Cond. Number MWGhosts Ghosts Protein name Q53EP0 132803.2 302% 2% Calumenin P5227277464.3 262% 2% Nucleophosmin P12956 69799.2 248% 1% Cytochrome coxidase subunit 2 O60716 108103.3 220% 4% Glutaminase kidney isoformP05121 45031.1 218% 5% Integrin beta-5 P18621 21383.3 218% 13% Plasmamembrane calcium-transporting ATPase 1 Q9H845 68716.8 209% 7% NucleolinQ14103 38410.3 198% 0% 60S acidic ribosomal protein P0 P61916 16559.5195% 2% Atlastin-3 Q02978 34039.9 193% 3% NADH dehydrogenase[ubiquinone] iron-sulfur protein 8 Q969X5 32571.5 186% 8% Neutralalpha-glucosidase AB P07858 37796.8 179% 2% Cytochrome c oxidase subunit4 isoform 1 O95831 66859 176% 4% Complement component 1 Qsubcomponent-binding protein P51148 23467.8 161% 6% 3-hydroxyacyl-CoAdehydrogenase type-2 Q9H0U4 22157.2 161% 1% AP-2 complex subunit alpha-1O75390 51679.6 157% 3% Adenylyl cyclase-associated protein 1 Q8TCJ293613.8 155% 1% Collagen alpha-1(III) chain P21964 30017.6 148% 0%Mitochondrial carrier homolog 2 O43852 37083.6 143% 2% Fibronectin typeIII domain-containing protein 3B O95782 107477.9 142% 5% Ras-relatedprotein Rab-1B P50416 88310.8 142% 2% Alpha-actinin-1 Q9NYU2 177077.4140% 1% Hydroxymethylglutaryl-CoA lyase P46977 80476.9 138% 3% 40Sribosomal protein S12 P49821 50784.9 137% 5% Actin-related protein 3Q12797 85809.5 135% 5% 40S ribosomal protein S20 P46940 189132.9 133% 1%Thy-1 membrane glycoprotein Q9ULV4 53215.1 131% 3% Coronin-1C O00159121648.1 129% 4% NADH dehydrogenase [ubiquinone] 1 alpha subcomplexsubunit 10 P07996 129299.2 129% 2% Mitochondrial-processing peptidasesubunit beta O14773 61209.7 129% 6% Mesencephalic astrocyte-derivedneurotrophic factor P10620 17587.2 128% 3% 60S ribosomal protein L8Q9NSE4 113719.1 121% 4% Peptidyl-prolyl cis-trans isomerase A O0046984631.8 120% 2% 39S ribosomal protein L49 P20340 23577.9 100% 100% 60Sribosomal protein L3 P80723 22680 100% 33% Endoplasmic reticulumresident protein 44 Q96AQ6 80594.2 100% 27% 40S ribosomal protein S7P08962 25619.1 100% 25% Prolyl 4-hydroxylase subunit alpha-2 P5028165842 100% 25% Transmembrane emp24 domain-containing protein 4 P629888559.6 100% 23% Electron transfer flavoprotein subunit alpha P2129120553.8 100% 21% Protein S100-A10 P62879 37307.1 100% 21% 40S ribosomalprotein S23 Q92499 82379.9 100% 15% Myosin-11 P05388 34251.8 100% 14%Heterogeneous nuclear ribonucleoprotein D0 O96000 20763.2 100% 12%Catenin beta-1 P23528 18490.7 100% 12% Seprase P15313 56797 100% 12%Translocation protein SEC62 Q9UBS4 40488.7 100% 12% Mitochondrial2-oxoglutarate/malate carrier protein P62917 28007.3 100% 12% Microsomalglutathione S-transferase 1 P00403 25548.2 100% 12% X-ray repaircross-complementing protein 6 P17813 70533.2 100% 11% Talin-1 P6135315787.8 100% 11% Mannosyl-oligosaccharide glucosidase Q00341 141368 100%11% Neutral cholesterol ester hydrolase 1 P0C7P4 30796.1 100% 11%Probable ATP-dependent RNA helicase DDX17 P49368 60495.4 100% 11%Cytochrome b-c1 complex subunit 7 Q9NQC3 129851.2 100% 10% 40S ribosomalprotein S15a Q9NX63 26136.2 100% 10% Interleukin enhancer-binding factor3 P84098 23451.3 100% 10% Protein ETHE1 Q12906 95279.2 100% 10%Coiled-coil-helix-coiled-coil-helix domain-containing protein 3 P1116654048.7 100% 9% Estradiol 17-beta-dehydrogenase 12 P06748 32554.9 100%9% Heterogeneous nuclear ribonucleoprotein M Q99584 11464.1 100% 9%Mitochondrial import receptor subunit TOM40 homolog P14927 13522 100% 8%T-complex protein 1 subunit gamma Q9UBI6 8001.2 100% 8% Pyruvatedehydrogenase E1 component subunit alpha P83731 17767.9 100% 8% Proteincanopy homolog 2 Q02218 115861.5 100% 8% Spectrin alpha chain P6102622526.6 100% 7% Filamin-A O95299 40725 100% 7% Myosin-Ic P22626 37406.7100% 7% Ras-related protein Rap-1A Q9UBG0 166548.2 100% 7% Succinyl-CoAligase [GDP-forming] subunit beta P07099 52915 100% 7% RNA-bindingprotein Raly O95182 12543.6 100% 7% Collagen alpha-2(VI) chain P0421617923.4 100% 7% Ras GTPase-activating-like protein IQGAP1 P62266 15797.7100% 7% Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2P35221 100008.6 100% 7% V-type proton ATPase subunit B Q13405 19186 100%7% Procollagen-lysine P18859 12579.6 100% 7% 60S ribosomal protein L32P25398 14505.5 100% 7% Dolichyl-diphosphooligosaccharide--proteinglycosyltransferase subunit STT3A O00571 73198.1 100% 7% Alpha-solubleNSF attachment protein P00367 61359.3 100% 7% ATPase family AAAdomain-containing protein 3A P62081 22113.3 100% 6% Pre-B-cell leukemiatranscription factor-interacting protein 1 Q15293 38866.2 100% 6%Delta-1-pyrroline-5-carboxylate synthase P21333 280561.4 100% 6%Ras-related protein Rab-10 P54920 33211.3 100% 6% ATP-dependent RNAhelicase DDX3X P62249 16435 100% 6% DnaJ homolog subfamily B member 11P62834 20973.7 100% 6% Heterogeneous nuclear ribonucleoproteins A2/B1O00264 21657.8 100% 6% Thioredoxin domain-containing protein 5 Q1671813450.2 100% 6% Calmodulin Q9Y3B3 25155.6 100% 6% 40S ribosomal proteinS27-like Q01518 51822.8 100% 6% Citrate synthase P35222 85442.3 100% 6%NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 Q9UKM932443.6 100% 6% Epoxide hydrolase 1 P51991 39570.5 100% 6% 60S ribosomalprotein L36 P04899 40425.1 100% 6% UPF0027 protein C22orf28 P62244 14830100% 6% Reticulon-4 P32322 33339.6 100% 5% Sarcoplasmic/endoplasmicreticulum calcium ATPase 1 P52815 21334.7 100% 5% Peptidyl-prolylcis-trans isomerase FKBP11 Q15019 41461.3 100% 5% Histone H2A type 1-AQ13011 35793.4 100% 5% Gelsolin P00558 44586.2 100% 5% ATP synthasesubunit g O75439 54331.6 100% 5% Thrombospondin-1 P43304 80801.7 100% 5%Histone H1.5 P18084 87996.2 100% 5% Plasminogen activator inhibitor 1Q7Z7H5 25926.4 100% 5% Matrix metalloproteinase-14 Q14554 59556.2 100%5% Peptidyl-prolyl cis-trans isomerase FKBP10 O60506 69559.6 100% 5%Enoyl-CoA hydratase Q9Y3U8 12245.9 100% 5% Heterogeneous nuclearribonucleoprotein A3 P62820 22663.4 100% 5% Fumarate hydratase Q13813284362.5 100% 5% 2-oxoglutarate dehydrogenase Q6P587 24826.7 100% 5%Interleukin enhancer-binding factor 2 O75964 11421.2 100% 5%Phosphoglycerate kinase 1 Q9Y2B0 20639.2 100% 4% 60S ribosomal proteinL24 Q12931 80059.8 100% 4% Myeloid-associated differentiation markerP51572 27974 100% 4% Alpha-actinin-4 Q5JRX3 117380.3 100% 4% Ornithineaminotransferase P36776 106422.5 100% 4% UPF0556 protein C19orf10 O9557127855.1 100% 4% 60S ribosomal protein L19 P61158 47341 100% 4% NADHdehydrogenase [ubiquinone] flavoprotein 1 Q9BS26 46941.5 100% 4% NADHdehydrogenase [ubiquinone] 1 alpha subcomplex subunit 7 P10515 68953.3100% 4% 40S ribosomal protein S2 Q71UM5 9470.9 100% 4% Transmembraneemp24 domain-containing protein 7 Q12905 43035.2 100% 4%Fumarylacetoacetate hydrolase domain-containing protein 1 P54886 87247.7100% 4% Reticulocalbin-1 P13073 19564.1 100% 4% Cathepsin B P1588031304.6 100% 4% Dihydrolipoyllysine-residue acetyltransferase componentof pyruvate dehydrogenase complex P60866 13364.3 100% 4%Aspartyl/asparaginyl beta-hydroxylase Q96I99 46481.5 100% 3% CatecholO-methyltransferase O96008 37869.2 100% 3% Protein S100-A13 P6131324131.1 100% 3% Alpha-aminoadipic semialdehyde dehydrogenase Q6PIU245778.8 100% 3% Vigilin Q8NBS9 47598.7 100% 3% Membrane-associatedprogesterone receptor component 1 Q14165 32213.6 100% 3% Galectin-1P04181 48504.3 100% 3% Presequence protease Q9NVI7 71324.8 100% 3%Glutamate dehydrogenase 1 Q92896 134463.3 100% 3% Pyruvate dehydrogenaseE1 component subunit beta Q99653 22442.4 100% 3% Profilin-1 P5470931492.1 100% 3% Serine beta-lactamase-like protein LACTB Q92520 24664.6100% 3% 40S ribosomal protein S17 P53597 36226.9 100% 3% 3-ketoacyl-CoAthiolase P15311 69369.8 100% 3% Myosin light polypeptide 6 P1380435057.6 100% 3% Ubiquitin O00217 23689.6 100% 3% 40S ribosomal proteinS16 Q9UIJ7 25549.6 100% 3% Microsomal glutathione S-transferase 3 P6215816826.8 100% 3% NADH dehydrogenase [ubiquinone] 1 alpha subcomplexsubunit 5 Q969H8 18783.3 100% 3% Lon protease homolog P83111 60655.1100% 3% Sodium/potassium-transporting ATPase subunit beta-3 Q1288487656.8 100% 3% Cofilin-1 P07954 54602.2 100% 3% Ras-related proteinRab-1A Q14697 106806.8 100% 3% Endoplasmic reticulum-Golgi intermediatecompartment protein 1 O14983 110181.8 100% 2% Pyrroline-5-carboxylatereductase 1 Q99714 26906.1 100% 2% Ras-related protein Rab-5C P20020138667.9 100% 2% 60S ribosomal protein L17 P62910 15849.8 100% 2% ATPsynthase-coupling factor 6 P39023 46079.8 100% 2% Ras-related proteinRab-6A Q9NYL4 22166.3 100% 2% 39S ribosomal protein L12 O14880 16505.6100% 2% GTP:AMP phosphotransferase mitochondrial Q92841 72326 100% 2%Putative cytochrome b-c1 complex subunit Rieske-like protein 1 Q96QV614224.9 100% 2% Septin-2 Q9Y224 28050.7 100% 2% Catenin alpha-1 P5580956122 100% 2% Transmembrane emp24 domain-containing protein 2 Q1372491860.9 100% 1% 60S ribosomal protein L27 Q6DD88 60503.5 100% 1%Epididymal secretory protein E1 Q96AY3 64204.3 100% 1% Proteindisulfide-isomerase A5 Q9Y6C9 33308.9 100% 1% C-type mannose receptor 2O15460 60863.7 100% 1% CD63 antigen Q53GQ0 34302.2 100% 1% Solutecarrier family 2 Q07021 31342.6 100% 1% Apoptosis-inducing factor 1P60660 16919.1 100% 0% Ezrin P35749 227197.9 100% 0% ATP-dependent RNAhelicase DDX1 P08559 43267.7 100% 0% Guanine nucleotide-binding proteinG(I)/G(S)/G(O) subunit gamma-12 O15260 30373.8 100% 0% Tropomyosinalpha-1 chain P16401 22566.5 100% 0% Glycerol-3-phosphate dehydrogenaseP11177 39208.1 82% 4% Golgi apparatus protein 1 P12110 108511.9 81% 3%Brain acid soluble protein 1 P42765 41897.7 79% 2% Succinyl-CoA ligase[GDP-forming] subunit alpha O94925 73414 76% 3% Catenin delta-1 Q9Y3I055175 72% 8% Guanine nucleotide-binding protein G(i) subunit alpha-2P55145 20243.6 71% 2% Tripeptidyl-peptidase 1 O43707 104788.5 71% 4%B-cell receptor-associated protein 31 P12814 102992.7 70% 4% CarnitineO-palmitoyltransferase 1 P49419 58450.2 66% 3% 60S ribosomal protein L15P09493 32688.7 66% 27% Surfeit locus protein 4 P35914 34337.8 62% 8%UDP-glucose:glycoprotein glucosyltransferase 1 P37802 22377.2 61% 4% 60Sribosomal protein L23a P07737 15044.6 57% 7% Calcium-binding protein p22P08708 15540.4 57% 5% Protein FAM3C P30084 31367.1 57% 3% Heterogeneousnuclear ribonucleoprotein Q P62937 18000.9 54% 9% Isoleucyl-tRNAsynthetase P19338 76568.5 50% 42% Acyl-CoA dehydrogenase family member 9P09382 14706.2 45% 28% Malectin P06396 85644.3 36% 2% Delta(3 P02461138479.2 35% 6% Dolichyl-diphosphooligosaccharide--proteinglycosyltransferase subunit STT3B Q9Y490 269596.3 34% 5% Endoglin P6275017684.1 24% 31% Transgelin-2

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

REFERENCE Other References are Cited Throughout the Application

-   1. Petersson, B. The dry mass of the pancreatic B-cells in relation    to their content of secretion granules. The Histochemical Journal 1,    55-58 (1968).-   2. Loewenstein, J. E. & Cohen, A. I. Dry Mass, Lipid Content and    Protein Content of the Intact and Zona-Free Mouse Ovum. J Embryol    Exp Morphol 12, 113-121 (1964).-   3. Lapidus, R. G., Tiffany, C. W., Isaacs, J. T. & Slusher, B. S.    Prostate-specific membrane antigen (PSMA) enzyme activity is    elevated in prostate cancer cells. Prostate 45, 350-354 (2000).-   4. Hur, E. M. et al. LIME, a novel transmembrane adaptor protein,    associates with p56lck and mediates T cell activation. J Exp Med    198, 1463-1473 (2003).-   5. Lewis, L. A. et al. The Lck SH2 phosphotyrosine binding site is    critical for efficient TCR-induced processive tyrosine    phosphorylation of the zeta-chain and IL-2 production. J Immunol    159, 2292-2300 (1997).-   6. Crowther, M., Brown, N. J., Bishop, E. T. & Lewis, C. E.    Microenvironmental influence on macrophage regulation of    angiogenesis in wounds and malignant tumors. J Leukoc Biol 70,    478-490 (2001).-   7. Martínez-Palomo, A., G. H. Bourne, J. F. D. & Jeon, K. W. in    International Review of Cytology, Vol. Volume 29 29-75 (Academic    Press, 1970).-   8. Marquez, M. et al. Charge-dependent targeting: results in six    tumor cell lines. Anticancer Res 24, 1347-1351 (2004).-   9. Carter, H. B. & Coffey, D. S. Cell surface charge in predicting    metastatic potential of aspirated cells from the Dunning rat    prostatic adenocarcinoma model. J Urol 140, 173-175 (1988).-   10. Bjellqvist, B., Basse, B., Olsen, E. & Celis, J. E. Reference    points for comparisons of two-dimensional maps of proteins from    different human cell types defined in a pH scale where isoelectric    points correlate with polypeptide compositions. Electrophoresis 15,    529-539 (1994).-   11. Bjellqvist, B. et al. The focusing positions of polypeptides in    immobilized pH gradients can be predicted from their amino acid    sequences. Electrophoresis 14, 1023-1031 (1993).-   12. Link, A. J., Hays, L. G., Carmack, E. B. & Yates, J. R., 3rd    Identifying the major proteome components of Haemophilus influenzae    type-strain NCTC 8143. Electrophoresis 18, 1314-1334 (1997).-   13. Link, A. J., Robison, K. & Church, G. M. Comparing the predicted    and observed properties of proteins encoded in the genome of    Escherichia coli K-12. Electrophoresis 18, 1259-1313 (1997).-   14. Campbell, R. B. et al. Cationic charge determines the    distribution of liposomes between the vascular and extravascular    compartments of tumors. Cancer Res 62, 6831-6836 (2002).-   15. Krasnici, S. et al. Effect of the surface charge of liposomes on    their uptake by angiogenic tumor vessels. Int J Cancer 105, 561-567    (2003).-   16. Croyle, M. A. et al. PEGylation of a vesicular stomatitis virus    G pseudotyped lentivirus vector prevents inactivation in serum. J    Virol 78, 912-921 (2004).-   17. Immordino, M. L., Dosio, F. & Cattel, L. Stealth liposomes:    review of the basic science, rationale, and clinical applications,    existing and potential. Int J Nanomedicine 1, 297-315 (2006).-   18. Peeters, L., Sanders, N. N., Jones, A., Demeester, J. & De    Smedt, S. C. Post-pegylated lipoplexes are promising vehicles for    gene delivery in RPE cells. J Control Release 121, 208-217 (2007).-   19. Rivest, V. et al. Novel liposomal formulation for targeted gene    delivery. Pharm Res 24, 981-990 (2007).-   20. Degli-Esposti, M. A. et al. Cloning and characterization of    TRAIL-R3, a novel member of the emerging TRAIL receptor family. J    Exp Med 186, 1165-1170 (1997).-   21. Lee, H. O., Herndon, J. M., Barreiro, R., Griffith, T. S. &    Ferguson, T. A. TRAIL: a mechanism of tumor surveillance in an    immune privileged site. J Immunol 169, 4739-4744 (2002).-   22. LeBlanc, H. N. & Ashkenazi, A. Apo2L/TRAIL and its death and    decoy receptors. Cell Death Differ 10, 66-75 (2003).-   23. Walczak, H. et al. Tumoricidal activity of tumor necrosis    factor-related apoptosis-inducing ligand in vivo. Nat Med 5, 157-163    (1999).-   24. Rieger, J., Naumann, U., Glaser, T., Ashkenazi, A. & Weller, M.    APO2 ligand: a novel lethal weapon against malignant glioma? FEBS    Lett 427, 124-128 (1998).-   25. Wu, X., He, Y., Falo, L. D., Jr., Hui, K. M. & Huang, L.    Regression of human mammary adenocarcinoma by systemic    administration of a recombinant gene encoding the hFlex-TRAIL fusion    protein. Mol Ther 3, 368-374 (2001).-   26. Griffith, T. S., Anderson, R. D., Davidson, B. L.,    Williams, R. D. & Ratliff, T. L. Adenoviral-mediated transfer of the    TNF-related apoptosis-inducing ligand/Apo-2 ligand gene induces    tumor cell apoptosis. J Immunol 165, 2886-2894 (2000).

1. A composition-of matter comprising a liposome attached to, orencapsulating a pharmaceutical agent, said liposome being composed of awhole cell membrane fraction.
 2. The composition-of-matter of claim 1,wherein said cell is a human cell.
 3. The composition-of-matter of claim1, wherein a cell source for said whole cell membrane is selected fromthe group consisting of a stem cell, a primary cell, a cell-line, anon-tumorigenic cell, a cancer cell and an immune cell.
 4. Acomposition-of matter comprising a liposome composed of a whole cellmembrane fraction of a stem cell.
 5. The composition-of-matter of claim4, wherein said stem cell comprises a human mesenchymal stem cell.
 6. Acomposition-of matter comprising a liposome composed of a whole cellmembrane fraction of a primary human cell.
 7. A composition-of mattercomprising a liposome composed of a whole cell membrane fraction of anon-tumorigenic human cell.
 8. The composition-of matter of claim 4,wherein said cell membrane is genetically modified to express anexogenous protein.
 9. The composition-of matter of claim 8, wherein saidexogenous protein is selected from the group consisting of a cellmarker, a targeting moiety and said pharmaceutical agent.
 10. Thecomposition-of-matter of claim 4, wherein said liposome encapsulates, orattached to a pharmaceutical agent.
 11. The composition-of-matter ofclaim 1, wherein said pharmaceutical agent is a therapeutic agent. 12.The composition-of-matter of claim 11, being non-immunogenic in a humansubject.
 13. The composition-of-matter of claim 4, wherein a cell sourceof said whole cell membrane fraction comprises cells autologous to ahost subject.
 14. The composition-of-matter of claim 4, wherein a cellsource of said whole cell membrane fraction comprises cellsnon-autologous to a host subject.
 15. The composition-of-matter of claim11, wherein said pharmaceutical agent is a diagnostic agent. 16.(canceled)
 17. The composition-of-matter of claim 4, wherein saidliposome is attached to a synthetic polymer at an external surfacethereof.
 18. (canceled)
 19. The composition-of-matter of claim 4,wherein said liposome has a size range of 30-1000 nm.
 20. Thecomposition-of-matter of claim 4, wherein said liposome is furthercomposed of synthetic lipids.
 21. A method of producing liposomescomprising, (a) subjecting cells to hypotonic conditions, so as toobtain ruptured cell membranes and/or ghosts; and (b) homogenizing saidruptured cell membranes and/or ghosts to thereby produce liposomes. 22.The method of claim 21, wherein said homogenizing is effected by: (c)sonicating said ruptured cell membrane and/or ghosts; and optionally (d)extruding said ruptured membrane and/or ghosts through a matrix ofpre-determined porosity.
 23. The method of claim 21 further comprisingconjugating a synthetic polymer to said liposomes following step (c).24. A method of encapsulating a pharmaceutical agent in a liposome, themethod comprising making the liposomes according to the method of claim21 and adding the pharmaceutical agent prior to the step ofhomogenizing.
 25. A pharmaceutical composition comprising as an activeingredient the composition-of-matter of claim 1 and a pharmaceuticallyacceptable carrier.
 26. A method of delivering a pharmaceutical agent,the method comprising administering to a subject in need thereof thecomposition of matter of claim 1, thereby delivering the pharmaceuticalagent. 27-28. (canceled)